THE DETECTION OF
POISONS AND POWERFUL DRUGS
AUTENRiETH WARREN
LABORATORY MANUAL
FOR
The Detection of Poisons
AND
Powerful Drugs
BY
DR. WILHELM AUTENRIETH
PROFESSOR IN THE UNIVERSITY OF FREIBURG i. B.
AUTHORIZED TRANSLATION
BY
WILLIAM H. WARREN, Ph.D.
FIFTH AMERICAN EDITION
WITH 25 ILLUSTRATIONS
PHILADELPHIA
P. BLAKISTON'S SON & CO
1012 WALNUT STREET
3 * >
COPYRIGHT, 1921, BY P. BLAKISTON'S SON & Co.
AUTHOR'S PREFACE
Additional matter in "Detection of Poisons" has made the
fourth edition considerably larger than the third. The seven
chapters now comprised in the book have been entirely revised
but the first three chapters remain unchanged in arrangement.
Chapter I treats of poisons volatile with steam. Organic
poisons, especially the alkaloids, form the subject of Chapter II.
Hydrastine and veronal, introduced into this chapter for the
first time, have been incorporated into the Stas-Otto process.
Chapter III deals with metallic poisons.
The toxic substances included in Chapter IV find no place in
the three groups just mentioned. As they seldom appear in
toxicological examinations, their significance is theoretical
rather than practical. The following members of this group
have been introduced for the first time, namely, cantharidin,
cytisine, ergot, papaverine, pilocarpine, saponin substances,
solanine, thebaine, and the toxalbumins, ricin, abrin and crotin.
Chapter V- deals with special qualitative and quantitative
methods, such as the quantitative estimation of phosphorus in
phosphorated oils; the electrolytic detection and estimation of
arsenic; the biological test for arsenic; the destruction of organic
matter and detection of arsenic by A. Gautier and G. Locke-
mann; Karl Th. Morner's estimation of minute quantities of
arsenic; methods of estimating alkaloids by H. Matthes, H.
Thorns and A. H. Gordin. This chapter also includes A. J. J.
Vandevelde's estimation of the toxic action of organic com-
pounds by means of blood haemolysis.
Chapter VI takes up the estimation of alkaloids and other
active principles in raw materials (drugs) and in their prepara-
tions. Pharmacopceial as well as other estimations, such as
that of nicotine in tobacco, caffeine in tea, coffee, cola prepara-
tions, etc., pilocarpine in jaborandum leaves, piperine in pepper,
VI AUTHOR S PREFACE
solanine in potatoes, and theobromine in cacao and its prepara-
tions have been included. The author has endeavored to treat
these subjects as thoroughly as possible.
Chapter VII describes the methods employed in detecting
carbon monoxide -in blood, in recognizing blood itself in stains
and in differentiating human from animal blood.
The new edition, though more comprehensive in its scope
than the last, has lost nothing in clearness because of the rear-
rangement of subject-matter. Beginners will probably confine
their attention to the first three chapters. Students of phar-
macy will undoubtedly add Chapter VI which deals with drug
assaying. The other chapters are designed more especially for
those who wish to become better acquainted with toxicological
procedures.
Descriptions of syntheses of organic drugs such as acetanilide,
antipyrine, phenacetine, pyramidone, salicylic acid, sulphonal
and veronal allow the student to review the methods employed
in connection with laboratory work. Structural, formulae of
alkaloids and their cleavage-products have been given only
when they have been definitely determined or shown to be highly
probable. By introducing this specific information the author
hopes to stimulate the student's interest in alkaloidal chemistry
which has become so important within recent years.
More advanced students will find in fine print brief
statements about the poisonous action of the better known
physiologically active substances as well as their distribution
in and elimination from the human organism. Repeated
references to larger treatises upon toxicology, especially to
R. Robert's " Intoxikationen, " have been made. Numerous
citations of literature enable the student to consult original
articles for fuller information.
The translation of the third and fourth editions into English
and Spanish and the proposed translation of the fourth edition
into Italian indicate that colleagues in other countries have
favorably received this work.
WlLHELM AUTENRIETH.
FREIBURG IN BADEN.
TRANSLATOR'S PREFACE
The fourth English edition of Professor Autenrieth's " Auffin-
dung der Gifte" was a translation of the fourth completely
revised German edition. The present, or fifth, English
edition is also a translation of the same work, for a fifth Ger-
man edition so far as the writer is aware has not yet appeared.
The last English edition, though adhering closely to the text
of the German work, included a few subjects not found in the
latter. Among these may be mentioned a fuller discussion of
the ''normal arsenic" question and the quantitative estimation
of arsenic and antimony by the Gutzeit test. These subjects
have been retained in the present edition. Owing to the
prominence attained of late by wood (methyl) alcohol, due to
ignorant or criminally careless substitution of this intoxicant
for grain (ethyl) alcohol, this substance has been added to the
list of volatile poisons. Aside from minor corrections of the
text, the omission and correction of certain tests, the intro-
duction of a few new tests of recent appearance in the litera-
ture, and the expansion of the index to include authors as well
as subjects, no changes of importance have been made in the
last edition of this work.
WILLIAM H. WARREN.
SOMERVILLE, NEW JERSEY.
vii
CONTENTS
PAGE
Author's Preface v
Translator's Preface vii
Introduction i
CHAPTER I
TESTS FOR PHOSPHORUS AND OTHER POISONS VOLATILE WITH STEAM FROM
ACID SOLUTION
PHOSPHORUS 5
Scherer's test; Mitscherlich's test; Blondlot and Dusart's test;
(a) in the Fresenius-Neubauer apparatus, (b) in the Hilger-
Nattermann apparatus; Detection of phosphorous acid; Phosphorus
in phosphorated oils; Detection and quantitative estimation by
the Mitscherlich-Scherer method; Metabolism in phosphorus
poisoning.
FURTHER EXAMINATION OF THE DISTILLATE
HYDROCYANIC ACID 19
Physiological action; Preliminary test; Detection; Quantitative
estimation; Detection in presence of potassium ferrocyanide;
Mercuric cyanide; Mercuric cyanide in presence of potassium
ferrocyanide.
CARBOLIC ACID 26
Action and fate in the organism; Detection; Quantitative estima-
tions; i. Gravimetrically; 2. Volumetrically (Beckurts-Koppes-
chaar); 3. Volumetrically (J. Messinger-G. Vortmann); Estimation
in urine; Carbolic acid in presence of aniline.
CHLOROFORM 35
Behavior in the human organism; Distribution in the cadaver;
Detection; Quantitative estimation in cadaveric material.
CHLORAL HYDRATE 38
Detection; Action and fate in the human organism; Quantitative
estimation in blood and tissues.
lODOFORM 41
Detection.
NITROBENZENE 42
Toxic action; Detection.
ANILINE 44
Toxic action; Detection.
CARBON DISULPHIDE 46
Toxic action; Detection; Quantitative estimation of carbon disul-
phide vapor in air.
X CONTENTS
PAGE
ETHYL ALCOHOL 49
Fate in the human organism; Detection.
METHYL ALCOHOL 52
Physiological action; Detection.
ACETONE 55
Occurrence in urine; Detection; Acetone in presence of ethyl alcohol;
Detection in urine.
BITTER ALMOND WATER AND BENZALDEHYDE 57
SYNOPSIS or GROUP I (CHAPTER I) 59
CHAPTER II
DETECTION OF THOSE ORGANIC SUBSTANCES WHICH ARE NOT VOLATILE WITH
STEAM FEOM ACID SOLUTION
STAS-OTTO PROCESS 63
A. EXAMINATION OF ETHER EXTRACT OF THE AQUEOUS TARTARIC ACID
SOLUTION 63
PICROTOXIN 66
Detection in beer.
COLCHICIN 68
PICRIC Aero 70
Action and elimination; Detection.
ACETANILIDE 73
Action; Detection; Examination of acetanilide urine.
PHENACETINE ....-." 75
Preparation; Detection.
SALICYLIC ACID . . 77
Detection; Quantitative estimation; Detection in urine.
VERONAL 79
Preparation; Physiological action; Detection in urine.
ANTIPYRINE 82
Preparation; Detection in urine.
CAFFEINE 83
Fate in human metabolism; Detection.
B. EXAMINATION OF ETHER EXTRACT OF THE AQUEOUS ALKALINE SOLUTION 85
CONHNE . . . ' 89
NICOTINE. ...,.......;,.. 90
Physiological action; Reactions.
ANILINE 93
VERATRINE 93
Preparation of crystalline and water soluble veratrine; Constitution;
Reactions.
STRYCHNINE 96
Physiological action; Detection; Detection in presence of brucine.
BRUCINE 100
ATROPINE . IO 2
Constitution; Reactions.
CONTENTS XI
PAGE
HOMATROPINE 105
COCAINE 105
Constitution; Behavior in the animal organism; Detection.
PHYSOSTIGMINE no
CODEINE in
NARCOTINE 113
Constitution; Detection.
HYDRASTINE 116
Preparation; Constitution; Reactions.
QUININE 119
Constitution; Detection.
CAFFEINE 122
ANTIPYRINE 123
Detection in urine.
PYRAMIDONE 124
Preparation; Behavior in the organism; Detection.
C. EXAMINATION OF ETHER EXTRACT AND OF CHLOROFORM EXTRACT OF
THE SOLUTION ALKALINE WITH AMMONIA.
a. Ether Extract 126
APOMORPHINE 127
/3. Chloroform Extract 130
Preliminary test for morphine; Purification of crude morphine.
MORPHINE 131
Constitution; Detection; Behavior in the animal organism.
NARCEINE 138
Constitution; Reactions.
SYNOPSIS OF GROUP II (CHAPTER II) 140
CHAPTER III
EXAMINATION FOR METALLIC POISONS
DESTRUCTION OF ORGANIC MATTER 148
Fresenius-v. Babo Method 148
By free chloric acid 151
C. Mai's Method 152
PRECIPITATION WITH HYDROGEN SULPHIDE 152
METALLIC POISONS I: Examination of that portion of the hydrogen sul-
phide precipitate soluble in ammonia-ammonium sulphide.
ARSENIC 156
Marsh-Berzelius method; Fresenius-v. Babo method; Bettendorff's
test; Gutzeit's test.
ANTIMONY, TIN, COPPER 163
METALLIC POISONS II: Examination of that portion of the hydrogen sul-
phide precipitate insoluble in ammonium sulphide 165
MERCURY, LEAD, COPPER, BISMUTH, CADMIUM 165
METALLIC POISONS III: Examination for Chromium and Zinc 168
ZINC. . , 168
Xii CONTENTS
PAGE
CHROMIUM 169
METALLIC POISONS IV: Examination for Barium, Lead and Silver of the
insoluble residue left on treatment with potassium chlorate and
hydrochloric acid 17
SYNOPSIS OF GROUP III (CHAPTER III) 171
THE ACTION or HEAVY METALS . . 172
FATE, DISTRIBUTION AND ELIMINATION OF METALS IN THE BODY .... 1 73
NORMAL ARSENIC 174
CHAPTER IV
1. EXAMINATION FOR THOSE POISONS WHICH DO NOT BELONG IN THE THREE
MAIN GROUPS
MINERAL ACIDS
HYDROCHLORIC ACID 183
NITRIC ACID '...-. 184
SULPHURIC ACID 186
SULPHUROUS ACID 188
OXALIC ACID 189
Toxic action; Distribution in the organism; Detection.
DETECTION OF FREE ALKALIES
POTASSIUM HYDROXIDE, SODIUM HYDROXIDE, AMMONIA 192
DETECTION OF POTASSIUM CHLORATE, SANTONIN, SULPHONAL, TRIONAL
POTASSIUM CHLORATE 194
Toxic action; Detection; Quantitative estimation; Behavior during
putrefaction; Detection in meat.
S \NTONIN 198
Constitution; Behavior in the organism; Detection.
SULPHONAL 200
Preparation; Detection in urine; Detection of hsematoporphyrin
in urine.
TRIONAL 203
2. POWERFUL ORGANIC SUBSTANCES SELDOM OCCURRING IN TOXICOLOGICAL
EXAMINATIONS
CANTHARIDIN .;.'.-". 203
Constitution; Detection.
CYTISINE 205
Preparation; Toxic action; Detection.
DIGITALIS BODIES . 207
Digitonin, Digitoxin, Digitalinum verum.
ERGOT .
209
Alkaloids; Sclererythrin; Detection of ergot in flour; Detection
and estimation of the alkaloids.
OPIUM 212
Meconic acid; Meconin; Selenious-sulphuric acid reagent for opium
alkaloids.
CONTENTS Xlll
PAGE
PAPAVERINE 215
Constitution; Detection.
PILOCARPINE 217
PTOMAINES . 219
SAPONINS 220
Physiological action; Detection in foaming beverages, such as beer,
etc.; Detection of githagin in flour.
HAEMOLYSIS AND PHYSIOLOGICAL SALT SOLUTION 224
SOLANINE 225
Toxic action; Detection.
THEBAINE 227
Constitution; Detection.
TOXALBUMINS 228
Abrin, Ricin; Crotin; Coagulation of blood and defibrinated blood.
CHAPTER V
SPECIAL METHODS
QUANTITATIVE ESTIMATION OF PHOSPHORUS IN PHOSPHORATED OILS
i. W. Straub's method; 2. A. Frankel's and C. Stich's method. . . 231
SPECIAL METHODS FOR DETECTING ARSENIC 233
Separation of arsenic as arsenic trichloride . . t 233
Electrolytic detection of arsenic 233
Destruction of organic matter and detection of arsenic by A. Gautier ,
and G. Lockemann 234
Electrolytic estimation of minute quantities of arsenic by C. Mai
and H. Hurt 237
Quantitative estimation of arsenic and antimony by the Gutzeit
method . 240
Biological detection of arsenic by means of penicillium brevicaule 242
Detection of arsenic in organic arsenic compounds, i.e. Cacodylic
acid; Arrhenal; Atoxyl. Their detection in urine 245
Quantitative estimation of minute quantities of arsenic by Karl Th.
Morner 247
DETECTION OF SALICYLIC ACID IN FOODS AND BEVERAGES; IN WINE,
MEAT PRODUCTS, MILK 250
MALTOL 251
CHLORAL HYDRATE IN TOXICOLOGICAL ANALYSIS BY R. MAUCH. ... 251
ALKALOIDAL ESTIMATIONS 253
1. By the picrolonate method of H. Matthes 253
2. By precipitation with potassium bismuthous iodide and decom-
position of the precipitate with alkali hydroxide-carbonate by H.
Thorns 255
3. By the method of H. M. Gordin 257
Quantitative estimation of strychnine and quinine in presence of
each other 258
Estimation of the toxicity of chemical compounds by blood haemo-
lysis by A. J. J. Vandervelde 258
xiv CONTENTS
CHAPTER VI
QUANTITATIVE ESTIMATION OF ALKALOIDS AND OTHER ACTIVE PRINCIPLES IN
RAW MATERIALS AND IN THEIR PREPARATIONS
PAGE
ALKALOIDAL ESTIMATIONS OF DRUGS AND THEIR PHARMACEUTICAL PREPA-
RATIONS ACCORDING TO THE GERMAN PHARMACOPCEIA 260
Estimation of alkaloid in aconite root 261
Estimation of cantharidin in Spanish fly 263
Estimation of cinchona alkaloids 264
i. In cinchona bark; 2. In aqueous extract of cinchona and in
alcoholic extract of cinchona.
Estimation of quinine in mixtures of cinchona alkaloids by the
sulphate method 268
i. Cinchona bark; 2. Cinchona extract.
Estimation of colchicin in colchicum seeds and in colchicum corms 269
Estimation of alkaloid in pomegranate bark 270
Estimation of caffeine in coffee, tea, cola nuts and Guarana paste 271
i. C. C. Keller's method; 2. A. Hilger-A. Juckenack's method.
3. A. Hilger-H. Gockel's method; 4. Socolof-Trillich-Gockel
method; 5. E. Katz's method; 6. K. Dieterich's method.
Estimation of alkaloid in ipecacuanha root 277
Estimation of nicotine in tobacco 279
i. R. Kissling's method; 2. C. C. Keller's method; 3. J. Toth's
method.
Estimation of hydrastine in hydrastis extract 281
Estimation of berberine 282
Estimation of hydrastine by the picrolonate method of H. Matthes
and O. Rammstedt 282
i. In fluid extract of hydrastis; 2. In hydrastis root.
Estimation of morphine in opium and in its pharmaceutical
preparations 283
i. In opium; 2. In extract of opium; 3. In wine of opium and in
tincture of opium.
Estimation of pilocarpine in jaborandum leaves 286
i. G. Fromme's method; 2. H. Matthes and O. Rammstedt's
method.
Piperine and its estimation in pepper 287
i. J. Konig's method; 2. Cazeneuve and Caillot's method.
Estimation of santonin in wormseed . > 289
i. K. Thaeter's method; 2. J. Katz's method.
Estimation of solanine in potatoes 291
i. O. Schmiedeberg and G. Meyer's method; 2. F. v. Morgen-
stern's method.
Estimation of alkaloid in nux vomica and its preparations .... 293
C. C. Keller's method 293
Method of the German Pharmacopoeia 294
i. In nux vomica; 2. In extract of nux vomica; 3. In tincture
of nux vomica.
CONTENTS XV
PAGE
H. Matthes and O. Rammstedt's method. 296
i. In extract of mix vomica; 2. In tincture of nux vomica; 3.
In nux vomica.
Estimation of strychnine in mixtures of strychnine and brucine by
C. C. Keller H. M. Gordin 298
Estimation of theobromine and caffeine in cacao and in chocolate 298
Estimation of alkaloid in the leaves of atropa belladonna, hyo-
scyamus niger and datura strammonium 300
Estimation of alkaloid in extract of belladonna, according to the
German Pharmacopceia, in extract of hyoscyamus 301
Assay of officinal extracts by E. Merck 302
Extract of belladonna; Extract of cinchona; Extract of strychnine.
CHAPTER VII
DETECTION OF CARBON MONOXIDE BLOOD, BLOOD STAINS AND HUMAN BLOOD
1. Recognition of carbon monoxide blood 304
2. Detection of blood stains 307
Haematin 308
Spectroscopic detection of blood 310
Other tests for blood 312
Schonbein-van Deen's test; Vitali's procedure in this test;
Schaer's procedure; Aloin test.
3. Biological detection of human blood 314
APPENDIX
PREPARATION OF REAGENTS
A. General alkaloidal reagents 317
B. Special reagents and solutions 320
C. The indicator iodeosine 322
INDEX 323
INTRODUCTION
Nearly all the common poisons and drugs may be placed in
one of three groups. This classification, based upon the
chemical behavior of these substances during isolation from
mixtures, is as follows:
Group I. The members of this group, when heated, vola-
tilize without decomposition and distil with steam from an acid
solution. Yellow phosphorus, hydrocyanic acid, carbolic acid,
chloroform, chloral hydrate, iodoform, aniline, nitrobenzene,
carbon disulphide and alcohol (ethyl and methyl) are the
principal substances of this class.
Group II. The members of this group are non-volatile,
organic substances which do not distil with steam from an
acid solution. But hot alcohol containing tartaric acid will
extract them from extraneous matter. Alkaloids, many glu-
cosides and bitter principles, as well as certain synthetic or-
ganic drugs, like acetanilide, phenacetine, antipyrine, pyrami-
done, sulphonal and veronal comprise this group.
Group III. This group includes all poisonous metals.
In toxicological analysis, therefore, poisons are divided into
three groups, each of which has its own special methods of pro-
cedure. A few poisons like mineral acids, caustic alkalies,
oxalic acid and potassium chlorate cannot be conveniently
placed in these three groups owing to differences in solubility
and other peculiarities. Special tests for such substances
must be made with a separate portion of material. Chapter
IV contains a description of the methods used in identifying
these substances.
The material must be thoroughly mixed and divided into
three or four approximately equal portions, unless the analysis
is to be limited to the detection of a single well-defined sub-
stance. One portion is tested for non-volatile, organic sub-
stances (Chapter II). The second portion is examined for
1
2 INTRODUCTION
volatile poisons (Chapter I) and the residue from this portion
is used in testing for poisonous metals (Chapter III). The
third portion is tested for substances considered in Chapter IV.
The fourth portion is held in reserve in case additional material
is needed to verify a doubtful result, or to replace a portion
accidentally lost during analysis.
Occasionally it is advisable to depart from the general pro-
cedure and follow a special method, especially in detecting a
single poison, or in estimating it quantitatively. For instance,
pure ether would not be the best solvent to use in extracting
strychnine quantitatively from an alkaline solution. A mix-
ture of ether and chloroform, or better pure chloroform would
be preferable, since strychnine is more soluble in the latter
solvent than in pure ether. For the same reason chloroform
should be used in the quantitative extraction of caffeine or
antipyrine. When only a small quantity of material is avail-
able for analysis, tests for all three groups of poisons may be
made with the same portion. In this case after removal of
volatile poisons (Chapter I) the residue should be divided into
two unequal portions. The larger portion should be tested for
non- volatile, organic poisons (Chapter II). The smaller por-
tion together with the residue left after extracting non- volatile,
organic poisons should be tested for poisonous metals (Chapter
III). It is advisable, however, even in such a case to reserve a
portion of material for any contingencies.
Organs of the human body like liver, kidneys, spleen, heart,
brain, stomach or intestines with contents should be cut into
small pieces and then finely chopped before being examined
chemically. An organ should first be cut into small pieces with
sharp, clean scissors and then minced with a clean chopping
knife in a new wooden bowl, or a small meat machine, which
has been carefully cleaned, may be used. Material may be
held with nickel plated tongs while being cut with scissors.
Largely to eliminate the unpleasant odor of viscera and facilitate
bringing them by hashing into a condition most favorable to
the action of acids or solvents, it has been suggested that they
be cooled ( 6 to 10).
DETECTION OF POISONS
CHAPTER I
VOLATILE POISONS
Yellow Phosphorus and Other Poisons Volatile with Steam from Acid
Solution
Scherer's Test. This test should precede the distillation
described on page 18. The principle of the test is that moist
phosphorus vapor and silver nitrate form black silver phosphide
(AgsP), metallic silver, phosphoric and sometimes phosphorous
acid. Place the finely divided material in a small flask and
cover with water if a sufficient quantity is
not present. Cut a V-shaped slit in the
cork and place the latter loosely in the
mouth of the flask so that the two strips of
filter paper are freely suspended (Fig. i).
Moisten one strip with silver nitrate and
the other with lead acetate solution. 1
Warm gently upon the water-bath (40 to
50). 2 If the silver but not the lead paper
is darkened, yellow phosphorus may be
present. If both papers are darkened,
hydrogen sulphide also is present. In the
latter case yellow phosphorus may be pres-
ent with hydrogen sulphide. In absence of FIG
hydrogen sulphide, darkening of the silver
paper is not final proof of yellow phosphorus, for any volatile
organic substance having reducing properties, as formalde-
1 A more sensitive "lead paper" may be obtained by using alkaline lead solu-
tion prepared by adding excess of sodium hydroxide to the solution of a lead
salt whereby Pb(OH)(ONa) and Pb(ONa) 2 are formed.
2 Temperatures in this book are expressed in Centigrade degrees. Tr.
3
4 DETECTION OF POISONS
hyde (H.CHO), or formic acid (H.COOH), may give the
same result.
Scherer's test is of value in proving the absence rather than the presence of
yellow phosphorus. It is a good preliminary test, as it excludes phosphorus if
the silver paper is unchanged.
Distillation. Place a portion of finely divided and thoroughly
mixed material in a large round-bottom flask and add enough
distilled water for free distillation. Then add tartaric acid
solution drop by drop until the mixture is acid after thorough
shaking. Practice analyses 1 usually require 20 to 30 drops of
10 per cent, tartaric acid solution.
In examining animal material, as the stomach or intestines
and contents, or organs, like liver, spleen and kidneys, it is often
unnecessary to addmuch water because enough is usually pres-
ent. First chop the material in a wooden tray with a steel knife.
In a medico-legal analysis the tray should be new. A meat
machine which has been carefully cleaned may be used. Thin
the material with a little distilled water, acidify with dilute
tartaric or sulphuric acid and finally distil.
If Scherer's test is positive, begin distilling with the Mit-
scherlich apparatus (Fig. 2) ; but if negative, distil in the usual
way with the Liebig condenser (see page 18). The distillate
may contain :
Yellow phosphorus Aniline
Hydrocyanic acid Ethyl alcohol
Carbolic acid Methyl alcohol
Chloroform Acetone
Chloral hydrate Carbon disulphide
lodoform Benzaldehyde
Nitrobenzene Bitter almond water
1 Laboratory practice in detecting poisons may be given by mixing small quan-
tities (from 0.03 to 0.05 or o.i gram) of a poison with dry bread or biscuit crumbs,
meal or meat. Finely chopped organs (liver, kidney, spleen, etc.), sausage
meat, beer, wine or milk may be used. Drugs like morphine, codeine, quinine,
acetanilide, phenacetine, antipyrine, caffeine, santonin, sulphonal, veronal,
calomel, tartar emetic, subnitrate of bismuth, etc., may be mixed with powdered
cane- or milk-sugar. The last kind of practice is especially suitable for students
of pharmacy.
VOLATILE POISONS
YELLOW PHOSPHORUS
Mitscherlich Method' of Detecting Yellow Phosphorus
The principle of this method is that yellow phosphorus
volatilizes with steam and becomes luminous in contact with air.
The phosphorescence is best seen in a dark room.
FIG. 2. Mitscherlich Apparatus.
Procedure. Arrange the apparatus as in Fig. 2. Support
the condenser in a vertical position and connect the upper end
with the flask by a glass tube about 8 mm. internal diameter.
This tube has two right-angle bends and each end passes through
6 DETECTION OF POISONS
a cork. Have condenser and tube scrupulously clean to avoid
interference with the phosphorescence.
Have the flask at most not more than a third full. This pre-
caution is necessary because many materials, containing protein
substances like albumin,, albumose, etc., and starchy matter,
when distilled in aqueous solution, cause more or less foaming
which is liable to carry over solid matter into the receiver. Use
as the receiver an Erlenmeyer flask containing a little distilled
water (3 to 5 cc.) into which the end of the condenser dips.
This precaution prevents loss of easily volatile substances like
hydrocyanic acid and chloroform. Heat the flask upon a wire
gauze of fine mesh, asbestos plate or sand-bath and bring the
contents to boiling by raising the temperature gradually.
There is some danger of burning or carbonizing organic matter
on the bottom of the flask, if heat is applied too strongly or
rapidly. When boiling begins, make the room as dark as pos-
sible and watch for phosphorescence in the tube and condenser.
It usually appears as a luminous ring or band in the upper part
of the condenser. When this is distinctly visible, the presence
of yellow phosphorus is established. Phosphorescence during
distillation with steam is very characteristic of yellow phos-
phorus and frequently is the only sure and unquestionable test
for this element.
Phosphorescence is a process of oxidation by which phos-
phorus vapor is changed to phosphorous acid. Should it not
appear immediately, distillation must be continued for some
time, since certain substances like ethyl or methyl alcohol, ether,
turpentine and many other ethereal oils either prevent the
phenomenon entirely or seriously retard it. Considerable
carbolic acid, creosote, chloroform, chloral hydiate, as well as
hydrogen sulphide, may completely prevent phosphorescence.
K. Polstorff and J. Mensching 1 have shown that mercuric
chloride as well as other mercury compounds may also interfere
with phosphorescence. Possibly mercuric chloride carried over
by steam is reduced to metallic mercury by phosphorus vapor.
In that case the metal should appear in the distillate. The
1 Berichte der Deutschen chemischen Gesellschaft 19, 1763 (1886).
VOLATILE POISONS 7
fact that both metallic mercury and phosphoric acid can be
detected in the distillate favors the supposition that action
takes place between phosphorus vapor and mercuric chloride.
Phosphorescence, however, often appears when these sub-
stances have passed over. But even when prolonged dis-
tillation fails to give a positive result, this must not be accepted
as final proof of the absence of phosphorus until other tests
have been made. Whatever the result, evaporate a portion
of the distillate to dryness on the water-bath with excess of
saturated chlorine water, or with a little fuming nitric acid.
Phosphorus always imparts a strong odor to the distillate.
Small drops of phosphorus appear if the quantity is large, and
the solution contains phosphorous acid. Dissolve the residue
from evaporation in 2 to 3 cc. of water and test in two separate
portions for phosphoric acid.
1. Ammonium Molybdate Test. Acidify the solution with
a few drops of concentrated nitric acid. Add an equal volume of
ammonium molybdate solution
and warm to about 40. Phos-
phoric acid precipitates yellow
ammonium phospho-molybdate.
2. Ammonium Magnesium
Phosphate Test. Add magnesia
mixture 1 to the second portion.
Phosphoric acid gives a white
crystalline precipitate of ammo-
nium magnesium phosphate (H 4 N)-
MgP0 4 .6H ! 0. Vigorous shaking FlG '
favors precipitation. When only
traces of phosphoric acid are present, long standing is necessary
before the precipitate appears. Always examine the precipi-
tate with the microscope. It should consist of well-formed
1 Magnesia mixture is a clear solution prepared by mixing equal volumes of
magnesium chloride, ammonium chloride and ammonium hydroxide (about
10 per cent.) solutions. It contains the readily soluble double chloride of ammo-
nium and magnesium which is not decomposed by ammonium hydroxide. This
reagent is prepared as needed and should be perfectly clear and colorless.
8 DETECTION OF POISONS
crystals or at least should be crystalline. These crystals are
transparent, acicular prisms (Fig. 3) .
Notes. A. Fischer 1 states that substances interfering more or less with the
detection- of phosphorus by the Mitscherlich method are usually less troublesome
if Hilger and Nattermann's procedure is used (see page 15). The essential
feature of the latter process consists in allowing steam charged with phosphorus
to pass into the air, or in admitting air into the apparatus.
Detection of Phosphorus and Phosphorous Acid
(Blondlot 2 -Dusart 3 )
When the Mitscherlich method fails to show phosphorus, it
is often necessary to test for phosphorous acid. This is the
first product in the oxidation of phosphorus and is easily formed.
The Blondlot-Dusart method shows the slightest trace of phos-
phorous (H 3 PO 3 ) and hypophosphorous (HsPC^) acids as
well as yellow phosphorus. The method consists in converting
yellow phosphorus into phosphine (PH 3 ) by nascent hydrogen.
The lower oxidation products of phosphorus, namely, hypo-
phosphorous (H 3 PO 2 ) and phosphorous (H 3 PO 3 ) acids, 4 when
warmed with zinc and dilute sulphuric acid are reduced to
phosphine by nascent hydrogen:
H 3 P0 2 + 4 H = PH 3 + 2H 2 O,
H 3 PO 3 + 6H = PHj. + 3 H 2 0. .
Phosphine, or hydrogen charged with phosphorus vapor,
burns with a characteristic green flame (Dusart's reaction) :
2 PH 3 + 4 2 = P 2 6 + 3 H 2 0.
The green flame is easily recognized by depressing a cold
porcelain dish or plate upon the flame. Detection of phosphorus
by the Blondlot-Dusart method depends upon these two facts.
A toxicological analysis usually deals with the detection of
traces of yellow phosphorus. Hydrogen after acting in the
nascent state upon the material is not directly examined for
1 Pflueger's Archiv, 97, 578 (1903).
2 Journal de Pharmacie et de Chimie (3), 40, 25.
8 Comptes rendus de 1'Academie des Sciences, 43, 1126.
4 Nascent hydrogen will not reduce ordinary, ortho-phosphoric acid (H 3 PO 4 ),
and its derivatives, pyrophosphoric (H 4 P 2 O 7 ) and meta-phosphoric (HPO 3 ) acids,
to phosphine.
VOLATILE POISONS 9
phosphorus but is first passed in to dilute silver nitrate solution.
Phosphorus and phosphine precipitate black silver phosphide 1
(Ag 3 P):
PH 3 + sAgNOs = Ag 3 P + 3 HN0 3 .
Thus traces of yellow phosphorus may be concentrated in the
silver precipitate from which nascent hydrogen will liberate
phosphine :
Ag 3 P + 3H = PH 3 + 3Ag.
If hydrogen produces a black or gray precipitate in the silver
solution, phosphorus is not necessarily present, as hydrogen
sulphide, arsine, stibine and reducing organic compounds be-
have similarly with silver nitrate. A black precipitate there-
fore should always be examined for phosphorus by the Dusart
reaction.
In the detection of yellow phosphorus, the Blondlot-Dusart
method combines two distinct operations, namely:
1. Preparation of the silver phosphide precipitate.
2. Examination of this precipitate in the Dusart apparatus.
Procedure, i. Preparation of Silver Phosphide. Thin the
finely divided material with water in a capacious flask where
hydrogen is being evolved from phosphorus-free zinc and pure
dilute sulphuric acid (1:5). In testing for phosphorous acid
alone (see page 14) use the filtrate from an aqueous extract
of the material, or the filtrate from the residue left after the
Mitscherlich distillation (see page 5). Nascent hydrogen
should act for 1.5 to 2 hours, or even longer, and pass through
neutral silver nitrate solution in the receiver at the end of the
apparatus. If yellow phosphorus is present, the hydrogen will
contain phosphorus and phosphine and cause a black precipi-
tate of silver phosphide in the silver nitrate solution. Collect
the precipitate upon a small ash-free paper, wash with a little
cold water and examine in the Dusart apparatus as described
elsewhere.
1 Phosphorus cannot be determined quantitatively as silver phosphide because
this compound is partially decomposed by water. Phosphoric and phosphorous
acids pass into solution:
(a) 2 A g3 P + sO + 3H 2 O = 6Ag + 2 H 3 PO 4 ,
(b) 2 Ag 3 P + 3 + 3 H 2 = 6Ag + 2 H 3 P0 3 .
10
DETECTION OF POISONS
If there is silver phosphide in the precipitate, the filtrate
will contain phosphoric or phosphorous acid (see Note, page 9).
To detect phosphoric acid, first add hydrochloric acid to remove
excess of silver from this filtrate. Filter through paper pre-
viously well washed with acid and water and completely expel
hydrochloric acid from the filtrate by evaporation upon the
water-bath with concentrated nitric acid. Dissolve the resi-
due in a little warm water and test for phosphoric acid with
ammonium molybdate or magnesia mixture.
2. Examination of the Silver Precipitate (AgsP) for Phos-
phorus. Two forms of apparatus may be used for this purpose,
namely:
(a) Fresenius-Neubauer 1 Apparatus. Generate hydrogen
in flask A (Fig. 4) from pure phosphorus-free zinc and dilute
FIG. 4. Fresenius-Neubaxier Apparatus.
sulphuric acid. Fill U-tube C with pieces of pumice stone
saturated with concentrated potassium hydroxide solution to
absorb any hydrogen sulphide. Use hard glass for tube D and
1 C. R. Fresenius, Qualitative chemische Analyse, XVI edition, page 521.
VOLATILE POISONS 11
have the tip F of platinum. The part marked E 1 is a glass
stop-cock or screw-tap. Reservoir B serves to hold liquid from
A when cock E is closed. A platinum tip is essential, other-
wise the flame instead of being colorless will always be yellow
from sodium in the glass. The place where the platinum tip is
fused into the glass should be cooled by wrapping cotton around
the glass and keeping it moist.
Procedure. Open E and let hydrogen from A pass for some
time through the apparatus to expel air. Then close E and
liquid in A will rise into B. Now open E just enough to allow
hydrogen to burn with a small flame which should be colorless
in the dark. If there is no trace of green in the inner cone and a
porcelain dish depressed upon the flame does not show an emer-
ald-green coloration, hydrogen is phosphorus-free. It is well
to repeat this test. To test the silver precipitate for phosphorus,
wash it with the paper into B with a little water.
When the entire precipitate is in A, close E until all the liquid
has risen from A into B. Then open E, light the hydrogen and
examine the flame in the dark. If the precipitate contains a
trace of silver phosphide, the inner cone will be green and a
porcelain dish depressed upon the flame will show an emerald-
green coloration. Have the hydrogen flame small so that its
color may be observed for some time.
(b) Hilger-Nattermann 2 Apparatus. Reduction takes place
in a 100 cc. flask closed by a rubber stopper with three holes,
two of which are for right-angle tubes just passing through the
stopper and the third for a thistle-tube going to the bottom of
the flask (Fig. 5). Hydrogen from a Kipp generator enters the
flask by one tube and leaves by the other. Attach to the latter
a U-tube filled with pieces of pumice stone saturated with
concentrated potassium hydroxide solution to absorb hydrogen
sulphide. Connect the other end of the U-tube with a hard
1 Fresenius and Neubauer use a screw pinch-cock instead of a gas-cock but by
means of a short rubber connector they interpose an ordinary cock between the
gas-flask A and the U-tube C.
2 Forschungsbericht iiber Lebensmittel und ihre Beziehungen zur Hygiene,
etc., 4, 241-258 (1897).
12 DETECTION OF POISONS
glass tube tipped with platinum. 1 Cut the paper containing
the precipitate into small pieces and place in the flask which
contains in addition a few pieces of phosphorus-free zinc and
enough water to seal the thistle-tube. Light the hydrogen after
it has passed through the apparatus for some time and been
found free from air by the usual test. Seen in the dark the
flame should be entirely colorless and burn without a green cone
or a greenish glow. 2 Hilger and Nattermann advise a spectro-
scopic examination of the flame to determine the purity of the
FIG. 5. Hilger- Nattermann Apparatus.
zinc. Pure zinc gives a hydrogen spectrum which shows only
an orange colored line in place of the yellow sodium line. The
minutest trace of phosphorus will give three green lines lying
to the right of the line D . The color of two of these lines is more
pronounced than that of the third. Having thus tested the
purity of zinc and sulphuric acid, pour a few cc. of dilute sul-
phuric acid (i : 5) through the thistle-tube into the flask con-
taining zinc and the silver precipitate. If the latter contains
phosphorus, the flame will show, though not always at once, a
green coloration which should be examined with the
spectroscope.
1 Hilger and Nattermann use a platinum tipped blow-pipe instead of a glass
tube tipped with the same metal. Cotton, which is kept moist and acts as
a cooler, is wrapped around the blow-pipe below the tip.
2 Zinc entirely free from phosphorus which will stand this test is difficult
to obtain.
VOLATILE POISONS 13
The Mitscherlich method affords a distillate especially suit-
able for the Blondlot-Dusart test. If this imparts a green color
to the hydrogen flame, there can be no question about the pres-
ence of phosphorus.
Although the Blondlot-Dusart test is very delicate, many
chemists refuse to accept it as a substitute for the Mitscherlich
test. Selmi states that animal material like brain, which con-
tains organic phosphorus compounds, yields after putrefaction
a distillate that often gives a black precipitate with silver ni-
trate solution. This will impart a green tinge to the hydrogen
flame in the Blondlot-Dusart test.
Z. Halasz,' however, has failed to confirm Selmi's results. He examined two
kinds of animal material by the Blondlot-Dusart method. First, he tested nor-
mal brains (man, calf, hog); second, the brain and other organs of rabbits that
had been given poisonous doses of phosphorus by the mouth and subcutaneously.
He examined these organs when fresh and also from week to week after more or
less pronounced putrefaction had set in, but could not detect phosphorus in the
brain in a single instance. These experiments disprove the earlier idea that
phosphorus normally present in the brain is so changed during putrefaction that
it can be detected by the Blondlot-Dusart reaction. He also failed to detect
phosphorus in the brain of rabbits poisoned by this element, though he found it in
other organs, as stomach and intestines, and in those rich in blood, as liver,
lungs and kidneys. He could always detect small or large quantities of phos-
phorus in any organ which this element had directly reached, or by which it had
been indirectly absorbed. If any compound containing phosphorus is really
formed in the brain during putrefaction, Halasz concluded that it is not volatile
with steam and does not give the Blondlot-Dusart reaction. On the basis of
these experiments Halasz holds that the Blondlot-Dusart method of detecting
phosphorus is just as reliable for forensic purposes as that of Mitscherlich.
Procedure of Halasz in the Blondlot-Dusart Method
Make a thin mixture of very finely divided material and boiled water in a flat-
bottom flask where hydrogen is being generated from phosphorus-free zinc and
dilute sulphuric acid. Warm upon the water-bath and pass the gas through an
absorption-tube provided with several bulbs and containing neutral silver nitrate
solution. Concentrated sulphuric acid and a little platinic chloride may be added
toward the end to hasten the evolution of gas. Nascent hydrogen thus acts upon
phosphorus in the animal material for 2-2.5 hours. Finally wash the silver
precipitate carefully with water and transfer it with the paper to the Blondlot-
Dusart apparatus.
1 Zeitschrift fur anorganische Chemie 26, 438 (1901).
14 DETECTION OF POISONS
Detection of phosphorous Acid
The reduction of phosphorous acid to phosphine by zinc and dilute sulphuric
acid takes place very slowly. Hilger and Nattermann state that even a few-
milligrams require the action of nascent hydrogen for 10 to 14 days. Moreover
careful manipulation is necessary because silver phosphide is quite unstable.
Water decomposes this substance into metallic silver and phosphorous acid and
the nitric acid present oxidizes the latter to phosphoric acid. Therefore when
special attention must be given to phosphorous acid, Hilger and Nattermann
recommend examining the silver precipitate (presumably Ag 3 P) after 2 days, or
at most 3, for phosphorus by the Blondlot-Dusart method and the nitrate for
phosphoric acid (see page 10).
Detection of Phosphorus in Phosphorated Oils
1. Straub's 1 Test. If a phosphorated oil is placed on the
surface of copper sulphate solution, phosphorus will gradually
pass from the oil to the aqueous solution and first form black
copper phosphide. The latter, acting as a carrier of oxygen,
oxidizes phosphorus still in the oil to phosphoric acid which
dissolves in the water.
Shake 10 cc. of phosphorated oil in a test-tube with 5 cc. of i
per cent, copper sulphate solution. According to the amount of
phosphorus a black or light brown coloration will appear in the
emulsion at once, or in a few minutes, and at most after 2
hours. Phosphoric acid in the aqueous solution may be recog-
nized by ammonium molybdate. At least 0.0025 per cent, of
phosphorus may be detected in this way.
A practical, therapeutic application of this reaction may be made in acute phos-
phorus poisoning. Administration of copper sulphate solution may prevent
absorption of free phosphorus still in the gastro-intestinal tract.
2. The Mitscherlich test is also applicable to phosphorated
oils, even though the oil may contain only 0.0002 gram of
phosphorus in 100 grams. But phosphorescence will not
appear unless air is admitted into the tube from time to time.
Phosphorus in oils' cannot be determined quantitatively by
the distillation method, for not more than 36 to 41 per cent, of
1 W. Straub, Miinchener medizinische Wochenschrift 50, 1145; Archiv der
Pharmazie 241, 335 (1903); and Zeitschrift fiir anorganische Chemie 35, 460
(1903)-
VOLATILE POISONS
15
phosphorus will distil over. The quantitative method recom-
mended by Straub (see page 231) may be used in that case.
Detection and Quantitative Estimation of Phosphorus
(Mitscherlich-Scherer)
Acidify a weighed portion of material with dilute sulphuric
acid and add a little ferrous sulphate. Distil in a gentle stream
of carbon dioxide, using a large flask fitted with a two-hole
stopper. Expel air completely from the apparatus by carbon
dioxide before heating. Use as receiver a flask fitted with a
PIG. 6. Hilger-Nattermann Apparatus for Detecting and Quantitatively Esti-
mating Phosphorus.
two-hole stopper. Pass the end of the condenser through one
hole and connect the other with a U-tube containing silver
nitrate solution. Evaporate the distillate upon the water-
bath with strong bromine water, or with concentrated nitric
acid, to oxidize phosphorus or any phosphorous acid formed.
Dissolve the residue in a little water and precipitate phos-
16 DETECTION OF POISONS
phoric acid with magnesia mixture. Weigh the precipitate as
magnesium pyrophosphate, Mg 2 P207. Heat the contents of
the U-tube with concentrated nitric acid. Precipitate silver
as silver chloride and filter. Concentrate the filtrate by evapo-
ration and precipitate phosphoric acid with magnesia mixture
as before. Combine this precipitate with the other. In dis-
tillation some phosphorus separates as globules in the first
receiver and any carried over as vapor by carbon dioxide is re-
tained by silver nitrate solution. As the steam distillation of
phosphorus is very slow, the process should be carried on for at
least 3 hours. Hilger and Nattermann recommend the ap-
paratus in Fig. 6 not only for detecting phosphorus but for
estimating it quantitatively. Air may be mixed with phos-
phorus vapor by means of stop-cock K and the characteristic
phosphorescence will appear.
Remarks Upon the Mitscherlich Test. Hilger and Nattermann state that
0.00006 gram of yellow phosphorus is the smallest quantity that can be detected
by the Mitscherlich method. When 200 cc. of water containing 0.0003 gram of
phosphorus were distilled, there was brilliant phosphorescence for 5 minutes.
The degree of dilution seems to have no effect upon the result, at least not within
limits occurring in practice. Hydrogen sulphide, always present in putrefying
animal matter, has no apparent effect upon phosphorescence. Free phosphorus
can be detected in putrid organic matter even after the lapse of considerable time.
Putrefactive and digestive processes appear to prevent oxidation of phosphorus.
Dragendorff detected phosphorus in an exhumed body several weeks after death.
Neumann found free phosphorus in a human body fourteen days and Elvers
eight weeks after death.
When an acid aqueous solution is distilled in the Mitscherlich apparatus, the
flask residue always contains phosphoric (H 3 PO 4 ), phosphorous (H 3 PO 3 ) and
hypophosphorous (H 3 PO 2 ) acids and red phosphorus. Distillation of a solution
of 0.0644 gram of phosphorus gave only 71.33 per cent, in the distillate. The
residue contained:
Phosphorus as phosphoric acid (H 3 PC>4) 18.93 per cent.
Phosphorus as phosphorous acid (H 3 PO 3 ) 2.15 per cent.
Phosphorus as hypophosphorous acid (HsPC^) 4-27 per cent.
Phosphorus as red phosphorus 2 . 98 per cent.
28.33
Oxidation of phosphorus may be prevented by distilling in a current of carbon
dioxide as in the Mitscherlich-Scherer method (see page 15).
Metabolism in Phosphorus Poisoning. Phosphorus has a very poisonous
action upon the processes of metabolism. Present as a vapor in the blood and
tissue fluids, it retards normal oxidative processes occurring in the animal organ-
VOLATILE POISONS 17
ism'during metabolism. In phosphorus poisoning the usual course of chemical
metabolism is wholly changed. Fat instead of being oxidized is deposited in the
organs in large quantity (fatty degeneration of the liver). Different observers
believe there is formation of fat from protein. During phosphorus poisoning the
quantity of protein broken down is greatly increased. In human metabolism
this applies to protein in both food and tissues. Yet the needs of the organism
are not satisfied and the conclusion is that the changes are not as complete as in
normal protein metabolism. This increase in the breaking down of tissues in
phosphorus poisoning recalls similar changes which take place during respiration
in insufficient oxygen. Accompanying these abnormal processes are certain
nitrogenous and non-nitrogenous products of metabolism which either are not
normally formed in the organism or appear merely as intermediate steps in the
formation of the oxidative products of metabolism. Decomposition of the
protein molecule goes in part only as far as the amino-acids. Consequently in
phosphorus poisoning the urine almost always contains
CH 3 \
>CH - CH 2 -- CH(NH 2 ) - COOH Leucine (a-amino-isobutyl-acetic acid),
CH 3 /
/OH (i) Tyrosine (p-oxyphenyl-a-aminopropionic
C 6 H 4 < acid)
X CH 2 - CH(NH 2 ) - COOH (4)
CH 3 - CH(OH) - COOH Sarcolactic acid (dextro-lactic acid).
In acute phosphorus poisoning the following acids can be detected in the urine
in greatly increased quantity:
/OH (i)
CeH^ Para-oxyphenyl-acetic acid.
X CH 2 - COOH (4)
xOH (i) Para-oxyphenyl-propionic acid (hydro-
CeH^ para-cumaric acid).
X CH 2 - CH 2 - COOH (4)
S - CH 2 - CH(NH 2 ) - COOH
Cystine, | , has also been detected in phosphorus
S - CH 2 - CH(NH 2 ) - COOH urine.
In phosphorus poisoning there is a marked decrease in the urea-content of
the urine but a decided increase in total nitrogen. A considerable part of the
nitrogen, that is to say, 25 per cent, or more of the total nitrogen, appears to leave
the body as ammonia. The urine of adults usually contains from 2 to 5 per cent,
of the total nitrogen as ammonia. The increase of ammonia may have some con-
nection with the increase in formation of acid during phosphorus poisoning.
Peptone-like substances, the presence of which is attributed to profound dis-
turbance of metabolism, frequently appear in the urine in phosphorus poisoning.
Various observers believe there is no longer any doubt as to the appearance of
geniune peptonuria. A glycosuria may also appear, or be easily induced by a
diet rich in sugar. In accord with this observation is the fact that the liver of an
animal poisoned by phosphorus is without the power to change glucose of the
blood into glycogen and store up the latter. In phosphorus poisoning the
alkalinity of the blood rapidly diminishes owing to the increased formation of
acid. Since persons poisoned by phosphorus have icterus (jaundice), bile-pig-
ment, or at least urobilin, can be readily detected in the urine.
2
18
DETECTION OF POISONS
The amounts of oxygen and carbon dioxide, which the organism respectively
takes up and gives off, show a marked diminution during phosphorus poisoning.
Only 48 per cent, of carbon dioxide, as compared with 100 per cent, under normal
conditions, may be eliminated.
In brief the chief characteristics of phosphorus urine are a strong acid reac-
tion; presence of protein (peptone-like substances) ; and frequently occurrence
of the amino-acids mentioned above, as well as fat cylinders, cell detritus, free
fat globules and blood-corpuscles.
Further Examination of the Distillate
When phosphorescence has been distinctly observed in the
Mitscherlich apparatus, it is advisable to stop distillation and
change the Liebig condenser to its customary position. This
simpler method of distilling is shown in Fig. 7 and should al-
ways be used in toxicological analysis when there is no occasion
to test for phosphorus.
PIG. 7. Distillation with Liebig Condenser.
Since the several poisons appearing here are not equally
volatile with steam, it is best to collect the distillate in two or
three fractions. The first will contain most of the easily volatile
substances like hydrocyanic acid, chloroform, ethyl and methyl
alcohol, acetone, iodoform and nitrobenzene. The others
(second and third) will contain substances less easily volatile
with steam like carbolic acid, aniline, chloral hydrate and carbon
disulphide. This must not be understood to mean that the first
part of the distillate will be free from substances that volatilize
VOLATILE POISONS
with difficulty, and the latter part free from those that volatilize
easily. In the main such will be the separation, but either part
of the distillate may contain traces of substances which will
appear in larger quantity in the other part.
The proper procedure is to distil until 5 to 10 cc. of liquid
have been collected. Divide the distillate into several portions
and test for hydrocyanic acid, chloroform, ethyl and methyl
alcohol, acetone, and, if necessary, also for iodoform and nitro-
benzene. Use the second and third portions (10 to 20 cc.)
to test for carbolic acid, aniline, chloral hydrate and carbon
disulphide.
Several of these volatile substances have a characteristic
odor, which makes it possible to recognize them with great
certainty in the original material and especially in the distil-
late. First, test the distillate for each individual substance
by its most characteristic reaction. Test for hydrocyanic
acid by the Prussian blue or sulphocyanate reaction; for ethyl
alcohol, acetone and acetaldehyde by Lieben's reaction; for
methyl alcohol by one of the oxidation tests; for carbolic
acid and aniline by Millon's reaction; for chloroform, chloral
hydrate and iodoform by the phenylisocyanide reaction; for
aniline with calcium hypochlorite solution; and finally, for carbon
disulphide with lead acetate and potassium hydroxide solutions.
When there is reason to believe that a certain substance is
present, confirm the result by making other characteristic
tests. It is seldom necessary to examine the distillate for all
the members of the group.
HYDROCYANIC ACID, HCN
Physiological Action. In whatever way applied, hydrocyanic acid is absorbed,
even from the skin. So rapid is the absorption of this poison that there is
evidence of an intoxication after a few seconds, or a few minutes at most. Part of
the poison thus absorbed passes from the body unchanged by way of the lungs.
Another part, usually much less, is eliminated by the kidneys and passes into the
urine. Sweat also is said to contain hydrocyanic acid.
Most of the absorbed hydrocyanic acid, though variable in quantity, undergoes
chemical change within the organism whatever be the form of its chemical com-
bination. Hydrocyanic acid is supposed to combine with loosely bound sulphur
of proteins and form sulphocyanic acid (HSCN) which is not nearly as toxic as
hydrocyanic acid. (Antidote for hydrocyanic acid.) Hydrocyanic acid after the
20 DETECTION OF POISONS
manner of the cyanohydrin reaction 1 might combine chemically with carbohy-
drates of the blood and tissues. Finally, putrefactive changes as well as ferment
action within the cadaver might convert hydrocyanic acid into ammonium for-
mate. 2 The last statement may explain the disappearance of hydrocyanic acid
until only traces remain in the cadaver. Thus the possibility of making more
than an approximately quantitative determination of hydrocyanic acid taken
internally is precluded from the beginning. Yet there are instances where the
poison has been found in the human cadaver after 14 days, and even after
100 and 180 days. After 48 days the author obtained enough hydrocyanic acid
in the distillate from stomach and intestinal contents of a child 4^ years old to
give the Prussian blue test in three different portions of the distillate after 3
to 4 hours.
Undoubtedly hydrocyanic acid has a very poisonous effect upon ferments for
it kills certain vegetable and animal enzymes, or at least strongly retards their
action. This acid interferes particularly with the action of that enzyme which
causes transfer of oxygen from blood-corpuscles and thereby gives rise to oxida-
tive processes (oxidation ferment, "respiration ferment"). Careful experiments
in metabolism have shown that warm-blooded animals under the influence of
hydrocyanic acid take up less than the normal amount of oxygen and con-
sequently give off less carbon dioxide, even though relatively large quantities of
oxygen are administered artificially. R. Kobert (Intoxikationen) regards hydro-
cyanic acid poisoning as an internal asphyxiation of the organs in presence of an
excess of oxygen. The oxidative processes of the blood are checked and so little
oxygen is used that the venous blood becomes arterial, that is to say, contains a
large quantity of oxyhaemoglobin. As a result the color of the venous blood
is bright red. This change of venous to arterial blood seems to be permanent
in cold-blooded but usually only transitory in warm-blooded animals. The
appearance of lactic acid in the blood and urine is due to the disturbing influence
of the poison up'on the oxidative processes of the organism. The processes of
normal metabolism in warm-blooded animals finally oxidize lactic acid to car-
bon dioxide and water. Consequently the appearance of lactic acid in the blood
is very transitory and it is not found in the urine at all. The occurrence of lactic
acid in the blood and a decrease in its alkalinity are concurrent. As a result of
very deficient oxidation during hydrocyanic acid poisoning, dextrose not infre-
quently appears in the urine.
The blood therefore in hydrocyanic acid poisoning is characteristically changed.
Venous blood becomes bright red. And moreover blood which contains this acid
cannot liberate oxygen from hydrogen peroxide, that is to say it has lost its
catalytic power. 3 Such a compound as cyano-hajmoglobin appears to exist and
* R ~ \) + HCN = R ~ C ^ OH (R denotes an y radical)
OH 2 ,O
'H-CN =H-C/
O H 2 \Q - NH 4
J Hydrocyanic acid poisons platinum black just as it does blood ferments. Put
about 5 cc. of 3 per cent, hydrogen peroxide solution in each of two test-tubes.
Add to one i or 2 drops of hydrocyanic acid (about i per cent, solution) and to
both a trace of platinum black. Pure hydrogen peroxide at once gives off oxygen
vigorously, whereas that containing hydrocyanic acid does not
VOLATILE POISONS 21
its formation in the blood of a person poisoned by hydrocyanic acid would seem
probable, yet for some unknown reason the union of this acid with haemoglobin
takes place either not at all or only with great difficulty.
In a chemical examination for hydrocyanic acid and potas-
sium cyanide the contents of the stomach and intestines, or-
gans rich in blood as liver, brain and heart, the blood itself
and sometimes the urine are most important. Examine such
material at once for hydrocyanic acid which may be recognized
by its characteristic odor, provided putrefaction has not gone
too far.
Preliminary Test. A special test (Schonbein-Pagenstecher
reaction) for hydrocyanic acid should precede distillation.
Acidify a portion of the original material in a small flask with
tartaric acid solution. Then suspend in the flask (see Fig. i)
a strip of " guaiac-copper " paper 1 without letting it touch the
liquid. Gently warm the contents of the flask upon the water-
bath. Neither hydrocyanic acid nor potassium cyanide is
present, unless the paper is turned blue or bluish green. But
the only conclusion to be drawn from a positive test is that
hydrocyanic acid, or an easily decomposable cyanide, may be
present. Further conclusions should not be drawn from a
positive result, since other substances like ammonia, volatile
ammonium compounds, hydrochloric acid and especially oxi-
dizing agents like ozone, hydrogen dioxide, nitric acid and
chlorine will turn the paper blue. Consequently, though very
delicate, this test cannot be accepted as conclusive proof of the
presence of hydrocyanic acid.
Mechanism of the Reaction. Hydrocyanic acid has nothing directly to do
with this reaction. But it forms ozone with copper sulphate and that turns the
guaiaconic acid of guaiac resin blue. Cupric cyanide (a) is an intermediate
product which furnishes ozonized oxygen as shown in (/3) :
(a) CuSO 4 + 2HCN = Cu(CN) 2 + H 2 SO 4 ,
(0) 6Cu(CN) 2 + 3H 2 = 6CuCN + 6HCN + O 3 .
1 Prepare "guaiac-copper" paper by saturating strips of filter paper with
freshly prepared, 10 per cent, alcoholic tincture of resin of guaiac. Dry these
strips in air and moisten before using with very dilute aqueous copper sulphate
solution (1:1000).
22 DETECTION OF POISONS
The actual chemical examination for hydrocyanic acid is
made by adding tartaric acid solution to the finely divided
material and distilling as described (see page 18). This acid
volatilizes easily with steam and most of it will appear in the
first part of the distillate. Therefore use the first 5 or 10 cc. of
distillate for the tests. Note cautiously the odor of the distil-
late, which is characteristic, and then proceed as follows:
1. Prussian Blue Test. Add to the solution (distillate) a
little potassmm hydroxide solution; then i or 2 drops of fer-
rous sulphate solution and i drop of ferric chloride solution.
Shake well and warm gently. Finally acidify with dilute hy-
drochloric acid. If much hydrocyanic acid is present, a pre-
cipitate of Prussian blue will appear immediately. But if the
quantity is small, the solution will have merely a blue or bluish
green color. After a long time (10 to 12 hours) a flocculent
precipitate of Prussian blue will settle to the bottom of the test
tube. The limit of delicacy is 1:5, 000,000. 1
Mechanism of the Reaction. 2 Hydrocyanic acid and potassium hydroxide
form potassium cyanide which with ferrous sulphate produces ferrous cyanide
(a). The latter combines with more potassium cyanide, forming potassium
ferrocyanide (0) which with ferric chloride precipitates Prussian blue (y), the
ferric salt of hydroferrocyanic acid (H 4 Fe(CN 6 ).
() FeS0 4 + aKCN = Fe(<!N)) 2 + K 2 SO 4 ,
(0) Fe(CN), + 4 KCN = K 4 Fe(CN)6,
(7) 3K4Fe(CN) 6 + 4 FeCl 3 = Fe 4 [Fe(CN) 6 ] 3 + I2KC1.
Prussian blue will not appear in presence of alkalies, since they decompose it
as follows:
Fe 4 [Fe(CN) 6 ] 3 + r 2 KOH = 3 K 4 Fe(CN) + 4Fe(OH) s .
Consequently test the final mixture with blue litmus paper to make sure it is
acid.
2. Sulphocyanate Test Add to a portion of the distillate
3 or 4 drops of potassium hydroxide solution and then a little
J Link and.Mockel, Zeitschrift fur analytische Chemie 17, 455 (1878).
2 The reaction may be explained by saying that all the ferrous salt in presence
of much potassium hydroxide is precipitated as ferrous hydroxide (a). The
latter with potassium cyanide then forms ferrous cyanide (0) and finally potas-
sium ferrocyanide (7) :
+ 2 KOH = Fe(OH) 2 + K 2 SO 4 ,
Fe OR 2 + 2KCN = Fe(CN) 2 + 2 KOH
(7) Fe(CN) 2 + 4 KCN = K 4 Fe(CN) 6 .
ferric chloride potassium ferrocyanide in presence of hydrochloric acid
finally gives Prussian blue (see above).
VOLATILE POISONS 23
yellow ammonium sulphide solution. Evaporate to dryness
upon the water-bath. Dissolve the residue in a little water,
and acidify with dilute hydrochloric acid. Filter through
double paper to remove sulphur, and add to the filtrate 2 or 3
drops of ferric chloride solution. If the distillate contained
hydrocyanic acid, a reddish to blood-red color will appear.
This is due to ferric sulphocyanate. The limit of delicacy is
i : 4,000,000.
Mechanism of the Reaction. Hydrocyanic acid and potassium hydroxide
form potassium cyanide which takes sulphur from yellow ammonium sulphide
and becomes potassium sulphocyanate (a) . The latter with ferric chloride forms
ferric sulphocyanate (/3):
() KCN + (H 4 N),S, = KSCN + (H 4 N)S,_i,
08) 3KSCN + FeCU = Fe(SCN) 3 +
3. Vortmann's 1 Nitropmsside Test. Add to a portion of the
distillate a few drops of potassium nitrite solution; then 2 to 4
drops of ferric chloride solution and enough dilute sulphuric
acid to give a bright yellow color. Heat to boiling, add suf-
ficient ammonium hydroxide solution to remove excess of iron
and filter. Add to the filtrate i or 2 drops of very dilute am-
monium sulphide solution. If the solution contained hydro-
cyanic acid, a violet color will appear and pass through blue,
green and yellow. The limit of delicacy is i : 312,000.
Note. This test is the reverse of the nitroprusside test for hydrogen sulphide
and is due to conversion of hydrocyanic acid to potassium nitroprusside, KjFe-
(NO)(CN)s, which causes the color changes when ammonium sulphide is added.
Very small quantities of hydrocyanic acid give a bluish green to greenish yellow
color.
4. Silver Nitrate Test. Acidify a portion of distillate with
dilute nitric acid, and add silver nitrate solution in excess. If
hydrocyanic acid is present, a white, curdy precipitate of
silver cyanide (AgCN) will appear. Excess of ammonium
hydroxide solution will readily dissolve this precipitate. The
limit^of delicacy is i : 250,000.
When a dilute aqueous solution of hydrochloric acid is dis-
tilled, the acid does not pass into the distillate. The pre-
cipitate, therefore, caused by silver nitrate solution cannot
1 Monatshefte fur Chemie 7, 416 (1886).
24 DETECTION OF POISONS
possibly be silver chloride, because at first nothing but pure
water distils from a i per cent, or weaker solution of hydro-
chloric acid. To remove hydrochloric acid, if present, re-
distil the first distillate over borax. This will retain hydro-
chloric but not hydrocyanic acid. It is advisable to collect
upon a filter, wash and dry the precipitate caused by silver
nitrate solution. If silver cyanide is heated in a bulb-tube, it
will form metallic silver and cyanogen gas. The latter may be
recognized by its characteristic odor. 1 The reaction is:
2 AgCN = 2Ag + (CN)i.
5. Picric Acid Test Make a portion of distillate alkaline
with potassium hydroxide solution and heat gently (50 to
60) with a few drops of picric acid solution. If hydrocyanic
acid is present, the solution will become blood-red. This is due
to formation of potassium isopurpurate.
Note. This test is not as delicate, nor as characteristic of hydrocyanic acid
as the other tests. If hydrogen sulphide is present, as it frequently is in distillates
from animal matter, picric acid solution will produce a red color owing to forma-
tion of picramic acid.
6. Weehuizen's 2 Test. Add a few drops of phenolphthalin
dissolved in dilute sodium hydroxide solution and then a little
copper sulphate solution (i : 2000) to a portion of the distillate.
Even in a dilution of i : 500,000 hydrocyanic acid will produce
a red color due to oxidation of phenolphthalin to phenolphthalein.
Such oxidizing agents as hydrogen peroxide, nitric acid and ferric
chloride do not give this test. Paper first moistened with alka-
line phenolphthalin solution and then with very dilute copper sul-
phate solution may be used. These phenolphthalin-copper sul-
phate papers turn red even in air containing hydrocyanic acid.
Under the conditions of the test phenolphthalin is oxidized to phenolphthalein:
^C 6 H 4 OH /C C H 4 OH
C6H4 OH = C^-CeH, OH + H 2 O
O CO
Phenolphthalin Phenolphthalein
1 Owing to the very poisonous character of cyanogen gas, it is safer to ignite the
gas at the rrouth of the bulb-tube. Cyanogen gas burns with a purple flame. Tr.
"Pharmaceutisch Weekblad 42, 271; and Pharmaceutische Zentralhalle 46,
256 (1905).
VOLATILE POISONS 25
On the other hand the phthalein heated with an alkaline hydroxide and zinc dust
is reduced to the phthalin.
Quantitative Estimation of Hydrocyanic Acid
To determine hydrocyanic acid quantitatively, acidify a weighed portion of
material with dilute sulphuric or tartaric acid and distil. Determine the
quantity of hydrocyanic acid in the distillate either gravimetrically or volu-
metrically. If the former method is used, collect the precipitate of silver cyanide
upon a weighed filter, wash and dry at 100 to constant weight; or ignite the
precipitate in a weighed porcelain crucible, and determine the quantity of me-
tallic silver obtained. If hydrochloric acid is present in the distillate, redistil
once over borax. The distillate will then be free from hydrochloric acid.
Detection of Hydrocyanic Acid in Presence of Potassium Ferrocyanide
When material contains non-poisonous potassium ferrocyanide, hydrocyanic
acid will appear in the distillate from a solution acidified with tartaric acid. In
an experiment, where i per cent, potassium ferrocyanide solution was distilled
with 0.03 gram of tartaric acid, the distillate contained considerable hydrocyanic
acid. Carbon dioxide, passed into hot, aqueous potassium ferrocyanide solution,
will liberate hydrocyanic acid even at water-bath temperature (75). To test for
potassium ferrocyanide beforehand, shake some of the original material with
water and filter. Test the filtrate with ferric chloride solution and dilute hydro-
chloric acid. If there is a precipitate of Prussian blue, potassium ferrocyanide is
present. To detect free hydrocyanic acid, potassium or sodium cyanide 1 with
certainty, in presence of potassium ferrocyanide, add to the material acid sodium
carbonate in not too small quantity and distil. Even long distillation over free
flame by this method will liberate hydrocyanic acid only from simple cyanides
and not from potassium ferrocyanide.
Detection of Mercuric Cyanide
When an aqueous solution of mercuric cyanide, which is exceedingly poisonous,
is distilled with tartaric acid, the distillate will contain hydrocyanic acid only
when a large quantity of mercuric cyanide is present. Distillation of 100 cc. of
i per cent, aqueous mercuric cyanide solution yields a distillate which gives the
Prussian blue test distinctly. But, if the quantity of mercuric cyanide is less and
the solution very dilute (for example, 100 cc. of o.oi per cent, solution), there will
not be a trace of hydrocyanic acid in the distillate, even though the solution is
strongly acidified with tartaric acid. If, however, a few cc. of freshly prepared
hydrogen sulphide water are added and distillation is resumed, mercuric cyanide
will be completely decomposed and the distillate will contain hydrocyanic acid.
Detection of Mercuric Cyanide in Presence of Potassium Ferrocyanide
The method of detecting hydrocyanic acid from simple cyanides, in presence of
potassium ferrocyanide, is not applicable to mercuric cyanide. Long distillation,
even from saturated acid sodium carbonate solution, gives no trace of hydro-
cyanic acid. But distillation in presence of not too little acid sodium carbonate,
after addition of a few cc. of freshly prepared, saturated hydrogen sulphide solu-
1 Mercuric cyanide is an exception.
26 DETECTION OF POISONS
tion liberates hydrocyanic acid from mercuric cyanide but not from potassium
ferrocyanide. It is possible to detect hydrocyanic acid by this method, when
very little mercuric cyanide is mixed with considerable potassium ferrocyanide.
For example, o.oi gram of mercuric cyanide in 100 cc. of 10 per cent, potassium
ferrocyanide solution can be detected. If potassium ferrocyanide is distilled
directly with hydrogen sulphide without addition of acid sodium carbonate,
the distillate will contain considerable hydrocyanic acid.
CARBOLIC ACID
Action and Fate of Carbolic Acid in the Animal Body
Concentrated carbolic acid coagulates and destroys the constituents of the
human body, especially proteins and protoplasmic structures. It has therefore a
very strong caustic action. But its action is not merely local, for after absorption
OH it shows an affinity particularly for the central nervous system,
I brain and spinal cord. The first indications of this in animals are
. strong stimulation, increased irritability as in the case of strych-
HC CH nine and paralysis. In man the period of stimulation is very
I || slow in appearing. In chronic poisoning, after repeated small
HC CH d oses o f carbolic acid, degeneration of the kidneys and liver is a re-
J5 suit of absorption. The human organism absorbs carbolic acid
H very rapidly. Absorption from the skin, the gastro-intestinal
tract, abrasions and the respiratory organs takes place readily. In the human
organism the poison is converted by conjugation with sulphuric acid into phenyl-
sulphuric acid:
HO-SCvOH + HO-C 6 H 6 = HO-SO 2 -OC 6 H 5 + H 2 O.
When the quantity of carbolic acid is very large, it is also converted into phenyl
glycuronic acid by conjugation with glycuronic acid, HOOC-(CH.OH)4CHO.
Considerable carbolic acid is oxidized within the body to dihydroxy-benzenes,
namely pyrocatechol (C 6 H 4 (OH) 2 (i,2)) and h>droquinol (CeH^OHMi^))-
These enter into synthesis with sulphuric acid and appear in urine as ethereal
salts of sulphuric acid. The dark color of "carbolic urine" is largely due to
further oxidation of hydroquinol, whereby colored products (quinone?) are
formed. In carbolic acid poisoning, urine often has a pronounced dark color
(greenish to black). Urine in other cases is amber-yellow at first, but standing in
air gives it a deeper color. When carbolic acid poisoning is suspected, the urine
should be examined chemically. "Carbolic urine" differs from normal human
urine in being nearly free from sulphuric acid, 1 the so-called "preformed sul-
phuric acid." Consequently barium chloride solution, in presence of excess of
acetic acid, gives only a slight precipitate of barium sulphate or none at all.
Filter when there is a precipitate and warm the clear filtrate with a few cc. of
concentrated hydrochloric acid. An abundant precipitate of barium sulphate
will usually appear. The mineral acid decomposes phenyl-sulphuric acid into
phenol and sulphuric acid which is then precipitated. Normal human urine
1 This is sulphuric acid present in urine as sulphates. It is also termed "pre-
formed sulphuric acid," by which is meant that it enters the body as such. In
this respect it differs from "ethereal," or "conjugate" sulphuric acids, which
result from syntheses within the body.
VOLATILE POISONS 27
contains considerably more "sulphate sulphuric acid" (A sulphuric acid) than
"ethereal sulphuric acid " (B sulphuric acid). The average proportion between
the two being: A SO^B S(>4 = 10:1. Barium chloride solution, added to
normal urine in presence of acetic acid, produces a heavy precipitate of barium
sulphate.
Distribution of Carbolic Acid in the Human Body After Poisoning
C. Bischoff 1 examined organs, removed from a man who died 15 minutes after
taking 15 cc. of liquid carbolic acid, and found the poison distributed as stated in
the table below. The organs in this case were perfectly fresh. Only a small por-
tion of the stomach was received.
Weight Organ Phenol
242 grams Contents of stomach and intestine o. 171 gram
112 grams Blood 0.028 gram
1480 grams Liver 0.63 7 gram
322 grams Kidney 0.201 gram
1445 grams Brain 0.314 gram
Bischoff distilled with steam until the distillate gave no further precipitate with
bromine water. The results show how rapidly carbolic acid is absorbed, and how
soon it is distributed throughout the body.
E. Baumann 2 has published certain facts relating to the quantity of carbolic
acid formed during putrefaction of protein substances. Baumann states that he .
obtained from 100 grams of fresh pancreas and 100 grams of moist fibrin, mixed
with 250 cc. of water, after 6 days of putrefaction 0.073 to 0.078 gram of tri-
bromophenol, corresponding to 0.0208 to 0.022 gram of phenol.
Urine gives a distinct test for carbolic acid 15 minutes after the poison has
been taken by the mouth, or hypodermically. This shows how rapidly carbolic
acid is absorbed. Most of the carbolic acid absorbed is eliminated in 4 or 5
hours. Schaffer 3 found the quantity of conjugate sulphuric acid in urine to
increase in exact proportion to the quantity of carbolic acid taken.
Tests for the Detection of Carbolic Acid
Carbolic acid distils quite easily with steam. Yet, to remove
the last traces, long-continued distillation is necessary. If
fractional distillation is made, when carbolic acid is present,
this substance will appear in the first and second fractions and
even in the third. Usually carbolic acid can be recognized by
its peculiar odor. When much carbolic acid is present, the
distillate is milky. Colorless or reddish globules may be seen
1 Berichte der Deutschen chemischen Gesellschaft 16, 1337 (1883;.
2 Berichte der Deutschen chemischen Gesellschaft 10, 685 (1877) and Zeit-
schrift fur physiologische Chemie i, 61 (1877-78).
3 Journal fur praktische Chemie, Neue Folge 18, 282 (1878).
28 DETECTION OF POISONS
floating in the liquid. Excess of potassium or sodium hydroxide
solution will dissolve carbolic acid and render the distillate
perfectly clear. Pure, anhydrous carbolic acid melts at 40
to 42 and distils at 178 to 182.
Decomposition of protein substances produces phenol and
especially para-cresol in small quantity. Traces of phenols
can almost always be detected in distillates from animal
matter in an advanced stage of decomposition. Millon's
reagent, and usually bromine water, will give positive tests
with such distillates.
I. Millon's Test. Millon's reagent, 1 heated with a solution
containing only a trace of carbolic acid, produces a red color.
An aqueous solution containing only 20 mg. of carbolic acid,
diluted i : 100,000, will give a distinct red color. If the phenol
solution is not very dilute, the color will appear even in the cold.
Though a very delicate test, it is not characteristic of carbolic
acid, because several other aromatic compounds behave simi-
larly. This is true of derivatives of mon-acid phenols like the
three cresols, salicylic acid, 2 para-
hydroxy-benzoic acid, para-hydroxy-
phenyl-acetic acid, para-hydroxy-
phenyl-propionic acid (hydro-para-
cumaric acid 3 ) and tyrosine. Aniline
heated with Millon's reagent also
gives a dark red color.
2. Bromine Water Test. Excess
of bromine water produces a yellow-
ish white, crystalline precipitate, even
Wlth V ^ dllute C ^ O ]ic add SOlu r
of 1:20,000. tions. It is a very delicate test
for carbolic acid. Phenol diluted
i : 50,000 yields, after some time, a precipitate made up in
part of well-formed crystals (Fig. 8).
1 For the preparation of this reagent see page 321.
Traces of salicylic acid volatilize with steam, at least in such quantity that it
can be detected with Millon's reagent.
3 Para-hydroxy-phenyl-acetic acid and hydro-para-curnaric acid are formed in
the putrefaction of proteins but are not volatile with steam.
VOLATILE POISONS- 29
If there is a sufficient excess of bromine water to give the supernatant liquid a
brownish red color, the precipitate consists only of tribromophenyl hypobromite,
C 6 H 2 Br 4 O. R. Benedikt 1 regards this compound as a brom-phenoxy-tribromo-
benzene with the structure
OBr , whereas Thiele and Eichwede 2 have ascribed to it the structure
I O
A I
BrC CBr /\
BrC CBr
HC CH
\/- HC CH
C \/
Br C
Br 2
This reaction takes place so easily that carbolic acid may even be determined
quantitatively as this tetrabromo-derivative (see page 31). It melts at 132-
OH I34 with evolution of bromine and crystallizes as lemon-yellow
J, leaflets from alcohol-free chloroform or ligroin. Heated with
/ /?\ alcohol, acetone, xylene, or aqueous sulphurous acid, this com-
BrCe 1 2CBr pound loses bromine and changes at once to 2,4,6-tribromo-
I II phenol, melting at 93-94. Salicylic alcohol (saligenin), sali-
^/ CH Cylic aldeh y de > salic ytic acid and para-hydroxy-benzoic acid
C are converted quantitatively by an excess of saturated bromine
Br water even in the cold into tribromo-phenyl hypobromite.
3. Ferric Chloride Test. Very dilute ferric chloride solution,
added drop by drop, imparts a blue-violet color to aqueous
carbolic acid solutions. Addition of dilute hydrochloric or
sulphuric acid changes this color to yellow. This test is not as
delicate as i and 2. It is entirely negative in presence of min-
eral acids. The limit of delicacy is about i : 1000.
4. Hypochlorite Test. Add a few cc. of ammonium hydroxide
solution to a dilute, aqueous carbolic acid solution, and then
2 or 3 drops of freshly prepared calcium or sodium hypochlorite
solution. Gentle warming will produce a blue color. Very
dilute carbolic acid solutions after some time give only a green
to blue-green color. F. A. Fliickiger 3 allows bromine vapor
to come into contact with the phenol solution which has been
mixed with a little ammonium hydroxide solution in a porcelain
dish.
1 Annalen der Chemie und Pharmazie 199, 127 (1879).
2 Berichte der Deutschen chemischen Gesellschaft 33, 637 (1900).
3 Pharmaceutische Chemie, page 287 (1879).
30 DETECTION OF POISONS
5. Nitrite Test Mix a carbolic acid solution with a dilute
alcoholic solution of ethyl nitrite, C 2 H 6 -O-N = O, 1 or iso-
amylnitrite, C 5 Hn-0-N = O, 2 and add concentrated sulphuric
acid from a pipette so that it forms a distinct under-layer. A
red zone will appear at the contact surface of the two liquids.
This is a very delicate test.
This test may also be made by adding the liquid under ex-
amination as an upper layer upon concentrated sulphuric acid
containing a trace of red fuming nitric acid.
6. H. Melzer's Benzaldehyde Test. 3 Add 2 cc. of concen-
trated sulphuric acid to i cc. of the solution (distillate) to be
tested for carbolic acid, then i or 2 drops of benzaldehyde and
heat. The mixture, at first yellowish brown, will become dark
red. At the same time a red resinous substance will appear,
unless the solution is too dilute. When cold add 10 cc. of water
and enough potassium hydroxide solution to give a distinct
alkaline reaction. If carbolic acid is present, a violet-blue
color will appear. To obtain this coloring-matter, acidify the
solution, extract with ether and evaporate the solvent. Alka-
lies, added to alcoholic solutions of the coloring-matter, produce
a blue color which acids discharge. This is a very delicate
test. One cubic centimeter of 0.05 per cent, carbolic' acid solu-
tion (= 0.0005 gram of carbolic acid), will still give the blue
color very distinctly.
Note. In absence of phenol concentrated sulphuric acid produces a dark
brown color with benzaldehyde. According to A. Russanow 4 the first condensa-
tion product between phenol and benzaldehyde in presence of concentrated
sulphuric acid is para-dihydroxy-triphenyl-methane which crystallizes in yellow-
ish needles:
C ' H6 \ ; HjC 6 H 4 -OH C 6 H 8V /C 6 H 4 -OH
w >C = i O + i = H 2 + >C< (i, 4).
H|C 6 H 4 -OH H / \C 6 H 4 -OH
Benzaldehyde " Phenol P-Dihydroxy-triphenyl-methane
Alkalies dissolve the pure crystals without color but, if these solutions are
exposed to air, oxidation takes place and a red or red-violet color appears. Prob-
l The officinal preparation is called "Spiritus Aetheris Nitrosi."
2 Amylium nitrosum of pharmacists.
3 Zeitschrift fur analytische Chemie 37, 345 (1898).
4 Berichte der Deutschen chemischen Gesellschaft 22, 1943 (1889).
VOLATILE POISONS 31
ably benzaurine, dihydroxy-triphenyl-carbinol, is first formed. This compound
is a brick-red crystalline powder soluble in alkalies with a violet color.
QUANTITATIVE METHODS OF ESTIMATING PHENOL
i. Gravimetric Estimation as Tribromophenol
The principle of this method is based on the complete pre-
cipitation of phenol from aqueous solution as tribromophenyl
hypobromite by an excess of saturated bromine water (see
Test 2) . The precipitate is practically insoluble in cold bromine
water and the results are very satisfactory. 1
Procedure. Place the aqueous phenol solution in a large
glass-stoppered flask. Add gradually, while shaking, saturated
bromine water until the supernatant liquid has a red-brown
color and bromine vapor is visible above the solution. Let
stand 2-4 hours and shake frequently. Then collect the pre-
cipitate in a weighed Gooch crucible and dry in a vacuum des-
iccator over sulphuric acid to constant weight. On the basis of
the following proportion calculate the weight of phenol corre-
sponding to the weight of the precipitate:
C 6 H 2 Br 4 O : C 6 H 5 -OH = Wt. of Ppt. found : x
409.86 94-05
Since the ratio -~r- = 0.2295, the weight of phenol may be
409.00
found by multiplying the weight of the precipitate by 0.2295.
2. Beckurts-Koppeschaar 2 Volumetric Method
Dilute sulphuric acid liberates hydrobromic acid from potas-
sium bromide (a) and bromic acid from potassium bromate (/3).
These two acids react according to (7) :
(a) KBr + H 2 SO 4 = KHSO 4 + HBr,
(ffl KBrO 3 + H 2 SO 4 = KHSO 4 + HBrO 3 ,
(7) S HBr + HBrOs = 3Br 2 + 3H 2 O.
1 The following results were obtained by F. Beuttel:
Phenol taken CeH 2 Br 4 O Phenol found Per cent, found
1. 0.103 grm. 0.4538 grm. 0.0997 g rn i. 96.2
2. 0.2072 grm. 0.8806 grm. 0.2014 grm. 98.6
3. 0.2072 grm. 0.8708 grm. 0.2006 grm. 98.6
2 Archiv der Pharmazie 24, 570 (1886).
32 DETECTION OF POISONS
Therefore addition of dilute sulphuric acid to a mixture of
potassium bromide and bromate solutions liberates bromine
which will convert phenol into a mixture of tribromophenol and
tribromophenyl hypobromite. The excess of free bromine
and also the loosely bound bromine atom of tribromophenyl
hypobromite will displace iodine from potassium iodide and
finally all the phenol will be present as tribromophenol:
C 6 H 2 Br 3 OBr + 2 KI = C 6 H 2 Br 3 OK + KBr + I 2
One molecule of phenol requires 6 atoms of bromine, as shown
by the equation:
5 KBr + KBrOs + 6H 2 SO 4 + C 6 H 8 OH = C 6 H 2 Br 3 OH + 3 HBr + 6KHSO 4 +
3H 2 0.
The following standard solutions are required:
1. o.oi n-potassium bromide solution, containing -
595.6
grams = = 5.956 grams KBr in 1000 cc.
2. o.oi n-potassium bromate solution, containing -
167.17
grams = - = 1.6717 grams KBr0 3 in 1000 cc.
3. o.i n-sodium thiosulphate solution, containing o.i
Na2S 2 O3.5H 2 O grams = 24.83 grams in 1000 cc.
4- Potassium iodide solution, containing 125 grams of KI
in 1000 cc.
Procedure. Put about 25 cc. of aqueous phenol solution
(distillate) into a flask having a tight glass stopper. Add 50
cc. each, of o.oi n-potassium bromide and o.oi n-potassium bro-
mate solutions, then 5 cc. of pure concentrated sulphuric acid
and shake vigorously for several minutes. The gradually
increasing opalescence of the solution becomes more and more
marked, as tribromophenol and tribromophenyl hypobromite
are precipitated. The yellow color which soon appears shows
excess of bromine. Open the flask in 15 minutes, add 10 cc.
of potassium iodide solution, shake and titrate free iodine in
5 minutes with o.i n-sodium thiosulphate solution.
VOLATILE POISONS 33
6 gram-atoms Br 6 X 79.96
Calculation. --- = - --^- = 4.7976 grams of bromine are
set free from a mixture of 1000 cc. of o.oi n-potassium bromide solution and 1000
cc. of o.oi n-potasssium bromate solution. A mixture therefore of 50 cc. of each
of the two solutions will give 0.2399 gram of bromine. This quantity of bro-
mine can convert 0.04704 gram of phenol into tribromophenol :
6Br : C 6 H 6 OH
479.76 94.05 = 0.2399 : x (x = 0.04704)
i cc. of o.i n-sodium thiosulphate solution corresponds to 0.012697 gram of
iodine and this quantity of iodine to 0.007996 gram of bromine. But 0.007996
gram of bromine will convert 0.00157 gram of phenol into tribromophenol:
6Br : C 6 H 5 OH
479.76 94.05 = 0.007996 : x (x = 0.00157)
Consequently, for each cc. of o.i n-sodium thiosulphate solution used, subtract
0.00157 from 0.04704 gram of phenol. This determines the quantity of car-
bolic acid in the 25 cc. of distillate taken.
3. Messinger-Vortmann 1 Volumetric Method
Excess of iodine (8 atoms of iodine to i molecule of phenol
dissolved in 4 molecules of potassium hydroxide) , added to an
alkaline phenol solution at 50-60, will produce a dark red, non-
crystalline precipitate. One molecule of phenol requires 6
atoms of iodine.
1. CH 6 OH + 3 I, = C 6 H 2 I 3 OH + 3 HI,
2. 3HI + 3 KOH = 3 H 2 + 3 KI.
This red precipitate dissolves in hot potassium hydroxide
solution with a red-brown color and appears as white 2, 4, 6-
tri-iodophenol, melting at 154-156, on addition of an excess of
dilute sulphuric acid. Messinger and Vortmann regard the
red compound as di-iodophenyl hypoiodite (C 6 H 3 I 2 OI) which
potassium hydroxide converts into the more stable isomeric tri-
iodophenol:
01 OH
#\ is converted by
1C CI potassium hy- 1C CI
droxide into
HC CH HC CH
H I
Red di-iodo- White 2. 4, 6-tri-
phenyl hypoiodite iodophenol
1 Berichte der Deutschen chemischen Gesellschaft 22, 2312 (1889); and 23,
2 753 (1890). See also Kossler and Penny, Zeitschrift fiir physiologische Chemie
17, 117 (1892).
34 DETECTION OF POISONS
Procedure. 1 The reaction between the alkaline phenol solu-
tion and iodine is rather slow in the cold but is hastened at 50
to 60.
Place a measured volume of aqueous phenol solution (5 to
10 cc.) in a small flask and add a measured volume of o.i
n-potassium hydroxide solution until the mixture is strongly
alkaline. Warm gently by dipping the flask in water at 60
and add 10-15 cc. more of o.i n-iodine solution than the
volume of o.i n-potassium hydroxide solution used, or until the
excess of iodine produces a strong yellow color. Agitation will
cause a deep red precipitate to appear. Cool the solution,
acidify with dilute sulphuric acid and dilute to a definite volume
(250 to 500 cc.). Filter an aliquot portion (100 cc.) rapidly and
determine excess of iodine with o.i n-sodium thiosulphate solu-
tion.
Calculation. Each molecule of phenol requires 6 atoms of
iodine. Therefore i atom of iodine = -~ = =
6 6
1 5-^75 phenol. 1000 cc. of o.i n-iodine solution, containing
o.i gram-atom of iodine, correspond therefore to 1.5675 grams
of phenol.
Note. This method will not give satisfactory results, unless
at least 3 molecules of sodium or potassium hydroxide are taken
for i molecule of phenol.
Estimation of Phenol in Urine
In determining carbolic acid in urine, the regular occurrence of phenols must
not be overlooked. After a mixed diet, the quantity of normal human urine
passed in 24 hours will yield approximately 0.03 gram of phenols (phenol and
more especially para-cresol) .
In certain diseases where there is excessive bacterial decomposition within the
organism, in the intestines for example, urine contains more of these phenols and,
consequently, more conjugate sulphuric acids. Even external application of
carbolic acid, for instance the use of carbolic acid water as a lotion, is sufficient to
i ncrease the quantity of phenyl-sulphuric acid in urine.
Detection of Carbolic Acid in Presence of Aniline
Aniline closely resembles carbolic acid in behavior toward Millon's reagent and
ne water. But the two substances can be easily separated. Add potas-
1 Use 0.5 to i per cent, carbolic acid solution for laboratory experiments.
VOLATILE POISONS 35
sium hydroxide solution in large excess and distil. The distillate will contain
aniline alone. Or make the solution strongly acid with dilute sulphuric acid,
and extract with ether which will dissolve only carbolic acid. Evaporate
the ether extract at a moderate temperature and examine the residue.
CHLOROFORM
Behavior in the Human Organism. When inhaled, chloroform first passes from
the air into the blood-plasma which then transmits it to the red blood-corpuscles
JT where it may accumulate in relatively large quantity. Air passed
through blood will remove chloroform completely. Pohl (see
Cl C Cl Robert's " Intoxikationen ") states that blood may contain 0.62
I per cent, of chloroform, three-fourths of which will be in the red
blood-corpuscles. At the height of a harmless narcosis the blood
contained only 0.035 per cent, of chloroform. Absorption of chloroform is rapid
from all parts of the body. The stimulative action of chloroform on the mucous
membranes of the respiratory passages explains such disturbances as coughing,
secretion of saliva and reflex slowing of respiration and heart-beat, occurring at
the beginning of narcosis. Dilatation of the blood-vessels of organs living after
death is due to paralysis caused by even small doses of chloroform. A drop in
blood-pressure accompanies paralysis of the brain and the heart's action is feebler
and slower. Several researches regarding the effect of inhaled chloroform upon
human and animal metabolism have shown an increase in the quantity of nitrogen
in the urine after prolonged narcosis because more protein is decomposed. The
amount of neutral sulphur and chlorine in the urine also increases. The increase
of the latter is due in part at least to the conversion of chloroform into chloride.
The acidity of the urine is also much higher. A final characteristic of chloroform
urine is the high content of reducing substances. The increased protein decom-
position in chloroform narcosis affects both reserve protein and that of the tissues.
This may explain degeneration in red blood-qorpuscles, glandular organs, the
heart, etc., after frequent narcoses or one of long duration.
The temporary or permanent paralysis of isolated animal or vegetable cells,
such as leucocytes, ciliated cells, yeast cells, algae and spores, is evidence of the
antiseptic action of chloroform when present in proper concentration in air or in a
liquid. This explains the use of i per cent, aqueous chloroform solution as an
antiseptic. Added to urine it acts as a preservative. Therefore it may be used
in the study of the action of enzymes but not of bacteria, though all micro-organ-
isms are not paralyzed or killed by chloroform water.
Pohl and Hans Meyer have studied the distribution of chloroform in the body
and found that the red blood-corpuscles and the brain are most likely to show this
poison. After chloroform has been inhaled, some will appear in the gastric
juice but at most only traces in the urine. In but two out of 15 cases of chloro-
form narcosis was this poison found in the urine and then only in traces.
Robert states that as a rule it is the exception to find chloroform itself in the
cadaver, because part of the poison is converted into chloride in the human organ-
ism and part is quickly exhaled during respiration. Usually it is possible to
detect chloroform in the breath of patients even 24 hours after narcosis. Bii-
dinger states that the mucus of the respiratory passages retains chloroform.
36 DETECTION OF POISONS
Tests for the Detection of Chloroform
Chloroform distils easily with steam and appears in the first
fraction in largest quantity. When much chloroform is present ,
it will separate from the distillate as heavy, colorless globules,
whereas a small quantity will remain in solution. This solution
usually has the characteristic odor and sweetish taste of chlo-
roform. The following tests should be applied to the first
fraction.
1. Phenylisocyanide Test. Add i or 2 drops of aniline to the
chloroform solution (distillate), and then a few cc. of aqueous,
or alcoholic potassium hydroxide solution. Gentle heat will
produce phenylisocyanide (CeHsNC). The penetrating and
very repulsive odor of this compound is easily recognized.
CHCls + C 6 H 5 .NH 2 + 3KOH = C 6 H S .NC + aKCl + 3H 2 O.
A. W. Hofmann states that this test will show with certainty
i part of chloroform in 5000 to 6000 parts of alcohol.
Note. Chloral, chloral hydrate, bromoform, iodoform and tetrachloro-
methane also give this test.
The fact that aniline boiled with potassium hydroxide solution gives a peculiar,
faintly ammoniacal odor, even when chloroform is absent, must not be over-
looked. There is small chance, however, of confusing this odor with the repulsive
smell of phenylisocyanide. In doubtful cases warm some water, containing a
drop of aniline and a trace of chloroform, with potassium hydroxide solution and
compare the odor with that in question.
2. Schwarz's Resorcinol Test. 1 Dissolve about o.i gram of
resorcinol ( C H dW)j|?y m 2 cc - of water > add a few dr P s of
sodium hydroxide solution and finally the liquid containing
chloroform. This mixture heated to boiling will develop even
in very dilute solution a yellowish red color attended by a beau-
tiful yellowish green fluorescence.
Chloral, bromal, bromoform and iodoform also give this test.
3- Lustgarten's 2 Naphthol Test. Dissolve a few centigrams
of a- or /3-naphthol hi i or 2 cc. of 33 per cent, aqueous potas-
sium hydroxide solution. Warm to 50 and add the solution to
1 Zeitschrift fur analytische Chemie, 27, 668.
2 Monatshefte fur Chemie, 3, 715 (1882).
VOLATILE POISONS 37
be tested. Chloroform will produce an evanescent blue color
which in contact with air will change to green and then to
brown. This color is less stable when /3-naphthol is used.
Acidification of the blue solution will precipitate naphthol col-
ored by a red dye-stuff. This precipitate is usually brick-red.
Chloral, bromal, bromoform and iodoform also give this test.
4. Fujiwara's Pyridine 1 Test. Mix 2 cc. of pyridine with
3 cc. of 10 per cent, sodium hydroxide solution, heat to boiling
and add i cc. of the liquid to be tested. Even a trace of chloro-
form will produce a bright, blue-red color.
Chloral, bromoform, iodoform and several other similar compounds also
respond to this test.
One part of chloroform in 1,000,000 parts of water, 500,000 parts of ether, or
300,000 parts of ethyl alcohol can be detected by this test. It is equally sen-
sitive toward the other substances mentioned.
5. Cyanide Test. Seal the liquid to be tested for chloroform
in a glass tube (pressure- tube) 2 with a little solid ammonium
chloride and alcoholic potassium hydroxide solution. Heat
for several hours in a boiling water-bath. Cool the tube, re-
move the solution and test for hydrocyanic acid by the Prussian
blue reaction. A positive test means that the distillate con-
tained chloroform. The following reactions take place:
(a) CHC1 3 + H 3 N + 3KOH = HCN + 3 KC1 + 3H 2 O,
(0) HCN + KOH = KCN + H 2 O.
6. Reduction Tests, (a) With Fehling's Solution. Warm
the liquid containing chloroform with Fehling's solution. A
red precipitate of cuprous oxide will appear.
(b) With Ammoniacal Silver Nitrate Solution. Add excess
of ammonium hydroxide to silver nitrate solution and then
the liquid containing chloroform. Heat will produce a black
precipitate of metallic silver.
These reactions are not characteristic of chloroform, because
many volatile organic substances, as formic acid and aldehydes
1 Chemical Abstracts n, 3201 (1917).
2 An ordinary citrate of magnesium bottle is a convenient apparatus for this
test. Wrap a towel around the bottle, place it in the water-bath and gradually
raise the temperature to boiling. Do not remove the bottle until it is cold. Tr.
38 DETECTION OF POISONS
which may occur in distillates from animal material, reduce
Fehling's and ammoniacal silver nitrate solutions.
Quantitative Estimation of Chloroform in Cadavers
(Ludwig-Fischer 1 )
Mix a weighed portion of material with water and distil
as long as there is any chloroform. To tell when this point is
Breached, apply the phenylisocyanide test to a few cc. of liquid
collected at the end of distillation. Add some calcium car-
bonate to combine with free hydrochloric acid. Warm the
distillate to about 60 and draw washed air through it by suc-
tion. Pass this air through a combustion-tube heated to high
temperature and then into silver nitrate solution acidified with
nitric acid. Weigh the precipitated AgCl (N).
Calculation :
3 AgCl : CHC1 3 = N : x.
This method is based upon the fact that chloroform heated
with steam above 200 is decomposed into carbon monoxide,
hydrochloric and formic acids:
(a) CHCl, + H 2 O = CO + 3 HC1.
(0) CHCU + 2H 2 O = H.COOH + 3 HC1.
In a series of blank experiments B. Fischer has shown that the stomach,
stomach contents and blood, of a person who has not taken chloroform, give
no volatile chlorine compounds under these conditions. By this method B.
Fischer found in the cadaver of a laborer, who had died during chloroform
narcosis, the following quantities of chloroform:
Weight Organ Chloroform
985 grams Stomach and contents and parts of
the intestine o.i gram
780 grams Lungs and blood from the heart o-55 gram
445 grams Portions of spleen, kidneys and liver traces
480 grams Brain 0.07 gram
From these results it appears that most of the chloroform was in the brain and
blood.
CHLORAL HYDRATE
Cl
C1_C_Q Chloral hydrate distils very slowly with steam from an acid
I solution. Therefore the complete distillation of a large quantity
H C OH of chloral hydrate requires considerable time. Chloral hydrate
I appears as such in the distillate.
1 Jahresbericht des chemischen Untersuchungsamtes der Stadt Breslau fur die
Zeit vom i April ^94 bis 31 Marz 1895.
VOLATILE POISONS 39
Tests for the Detection of Chloral Hydrate
Chloral hydrate like chloroform will give the phenyliso-
cyanide, resorcinol, naphthol and pyridine tests. But the
distillate containing chloral hydrate does not have the charac-
teristic chloroform odor which is also scarcely perceptible in
very dilute aqueous chloroform solutions.
Jaworowski 1 suggests the following tests to differentiate
chloral hydrate from chloroform:
1. Test with Nessler's Solution. Add a few drops of this re-
agent to an aqueous chloral hydrate solution and shake. It
will produce a yellowish red precipitate, the color of which will
change after a while to a dirty yellowish green. This is an
aldehyde reaction.
2. Test with Sodium Thiosulphate. Boil a few cc. of chloral
hydrate solution with 0.2-0.3 gram of solid sodium thiosulphate.
This will give a turbid liquid of brick-red color. A few drops
of potassium hydroxide solution will remove the turbidity and
change the color to brownish red.
When the quantity of chloral hydrate is not too small, it may
also be detected by the following procedure:
Decomposition of Chloral Hydrate. Heat a portion of the
distillate for 30 minutes under a reflux condenser with calcined
magnesium oxide (MgO) upon a boiling water-bath. Magne-
sium formate and chloroform are produced by decomposition
of chloral hydrate.
2 CC1 3 .CH(OH) 2 + MgO = aCHCl, + Mg(OOCH) 2 + H 2 O.
Proceed as follows to detect these products :
Chloroform. Distil a few cc. from the solution in the flask
and test for chloroform by the phenylisocyanide, resorcinol,
a-naphthol and pyridine tests.
Formic Acid. Filter the icsidue from the distillation, con-
centrate the nitrate to a few cc. by evaporation and divide into
two parts for the following reduction tests:
(a) Reduction of Mercuric to Mercurous Chloride. Add
a few drops of mercuric chloride solution and warm. Formic
1 Pharmaceutische Zeitung fiir Russland 33, 373, und Zeitschrift fur analytische
Chemie, 37, 60 (1898).
40. DETECTION OF POISONS
acid, if present, will produce a white precipitate of mercurous
chloride (calomel) :
Mg(OOCH) 2 + 4HgCl 2 = 2'Hg 2 Cl 2 + MgCl 2 + 2HC1 + 2CO 2 .
(b) Reduction of Silver Nitrate. Warmed with silver nitrate
solution, formic acid and its salts produce a black precipitate of
metallic silver:
Mg(OOCH) 2 + 4AgN0 3 = 4Ag + Mg(NO 3 ) 2 + 2HNO 3 + 2CO 2 .
Detection of Chloral Hydrate in Powders or Solutions
Extract a powder with cold water containing sulphuric acid,
filter, extract the filtrate several times with ether and spon-
taneously evaporate the ether extracts in a shallow dish or on a
clock-glass. Chloral hydrate imparts to the residue its char-
acteristic pungent odor. The odor of chloroform is easily
recognized by warming the residue with sodium hydroxide
solution:
CC1 3 -CH(OH) 2 + KOH = CHC1 3 + H.COOK + H 2 O
The phenylisocyanide, resorcinol, naphthol and pyridine
tests, as well as that with Nessler's reagent, should be applied
to the residue.
In the case of an aqueous solution of chloral hydrate, first
acidify with dilute sulphuric acid and repeatedly extract with
ether. Evaporate the ether extracts and examine the residue
as already described.
Note. Pure chloral hydrate forms transparent crystals which are dry, perma-
nent and colorless. This compound has a pungent odor, its taste being caustic
and faintly bitter. It dissolves with ease in water, alcohol and ether; and in 5
parts of chloroform. It melts at 58.
Action and Fate of Chloral Hydrate in the Human Organism
Applied locally chloral hydrate acts as a strong stimulant. Taken internally
it frequently stimulates the stomach. When it reaches the blood, it acts like
chloroform in paralyzing the brain, spinal cord and heart but usually no previous
stimulation is noticeable. There is marked decrease in blood-pressure due to
paralysis of the blood-vessels. Death from chloral hydrate poisoning is occa-
sioned by impaired circulation and respiration, in consequence of which the quan-
tity of oxygen taken in and of carbon dioxide given off is considerably diminished.
H. Meyer has shown that the narcotic action of chloral hydrate depends, as does
that of all compounds of the alcohol and chloroform group, upon the affinity of
the poison for lipoids, the fatty constituents of the nervous system. It is also
VOLATILE POISONS 41
held by the blood, especially by the red blood-corpuscles. Later it appears
unchanged, most abundantly in the cells of the brain and spinal cord (Kobert,
' ' Intoxikationen ") .
Only very little chloral hydrate taken internally passes as such into the urine.
As shown by v. Mering and Musculus, 1 the greater part by conjugation with gly-
curonic acid forms urochloralic acid (CsHnClsO?) which is eliminated as such in
the urine. This conjugated acid undergoes hydrolysis, when boiled with dilute
acids, and gives trichlor-ethyl alcohol and free dextro-rotatory glycuronic acid:
CsHnCUpT + H 2 = CC1 3 -CH 2 OH + HOOC-(CH.OH) 4 -CHO
Urochloralic Trichlor- Glycuronic
acid. ethyl alcohol. acid.
Urochloralic acid is therefore trichlor-ethyl glycuronic acid. It is crystalline
and with heat reduces silver solution as well as alkaline copper and bismuth so-
lutions. Consequently chloral urine behaves much like sugar urine but differs
from the latter in being strongly laevo-rotatory. The reduction of the aldehyde
chloral, to its corresponding primary alcohol, trichlor-ethyl alcohol, is especially
noteworthy as regards the behavior of chloral hydrate in the human organism.
Quantitative Estimation of Chloral Hydrate in Blood and Tissues
(Archangelsky 2 )
Distil the material for 12-20 hours with its own weight of 20 per cent, phos-
phoric acid, repeating the process if the distillate is turbid or yellow. To com-
plete the decomposition of chloral hydrate into chloroform and formic acid, add
50 cc. of sodium hydroxide solution to the distillate and concentrate on the water-
bath to about 20 cc. Neutralize the solution exactly and heat for 6 hours on the
water-bath with an excess of mercuric chloride solution. Finally weigh the
precipitated mercurous chloride. Satisfactory results were obtained by this
method when known quantities of chloral hydrate were added to blood and
organs. Using this method Archangelsky has shown that chloral hydrate is not
uniformly distributed in the blood but is contained especially in the blood-cor-
puscles. When narcosis begins there is less chloral hydrate in the brain than in
the blood. But later the percentage of the poison in the brain is higher than in
the blood. Archangelsky has further shown how much chloral hydrate the blood
must contain before narcosis can appear. A dog's blood must contain 0.03-0.05
per cent. When the blood contains 0.12 per cent., respiration ceases.
IODOFORM
lodoform crystallizes in shining hexagonal leaflets or plates. It may also
T appear as a rather fine crystalline powder, lemon-yellow in color and
having a penetrating odor somewhat like saffron. The melting-point
I C I of iodoform is approximately 120. It is nearly insoluble in water;
I soluble in 50 parts of cold and in about 10 parts of boiling alcohol;
and soluble in 6 parts of ether. It is also freely soluble in chloroform.
1 Berichte der Deutschen chemischen Gesellschaft 8, 662 (1875); and v. Mer-
ing, Ibid., 15, 1019 (1882).
2 Archiv fur experimentelle Pathologic und Pharmakologie, 46, 347 (1901).
42 DETECTION OF POISONS
Detection of lodoform
lodoform distils quite easily with steam and gives a milky
distillate having a characteristic odor. Extract this distillate
with ether and carefully test the residue left by the spontaneous
evaporation of the solvent. If much iodoform is present,
it will form yellow hexagonal plates. Dissolve the ether
residue in a little alcohol, and use this solution for the following
tests:
i. Lustgarten's 1 Test. Gently warm a few drops of alcoholic
iodoform solution in a test-tube with a little sodium phenolate
(CeHs.ONa) solution. 2 If iodoform is present, a red substance
will be deposited on the bottom of the tube. A few drops
of dilute alcohol will dissolve this precipitate with a carmine-red
color.
Also make the resorcinol, pyridine and phenylisocyanide
tests (see pages 36 and 37).
NITROBENZENE
Nitrobenzene has a strong poisonous action. Administration of very small
quantities of this compound has produced death in human beings. There are
J^Q^ records in the literature of several cases where 20 drops, and even
j 7 to 8 drops, have caused fatal results. But on the other hand
complete recovery has followed poisoning by much larger doses.
Fatal poisonings have come also from inhaling nitrobenzene vapor.
Within recent years nitrobenzene has been used to some extent as
HC CH an abortifacient. Nitrobenzene poisons the blood and changes its
\/ appearance. The blood has a chocolate color and at the same time
the red blood-corpuscles change their shape and go into solution.
As a result the blood is incapable of uniting with oxygen. The
blood of persons poisoned by nitrobenzene is said to contain less than i per
cent, of oxygen so that death is caused by asphyxiation. Healthy blood
contains about 17 per cent, of oxygen by volume. There seems to be no
methaemoglobin in blood containing nitrobenzene. Such blood examined
spectroscopically shows the two oxyruemoglobin bands and also a special
absorption-band between C and D (Fihlene's nitrobenzene band). It is proba-
ble that the slight solubility of this poison necessitates a definite incubation
period, for 2 to 3 hours usually elapse after nitrobenzene has been taken before
1 Monatshefte fur Chemie, 3, 715 ( Z 882).
2 Prepare sodium phenolate solution by mixing 20 grams of phenol with 40
grams of sodium hydroxide and 70 grams of water.
VOLATILE POISONS 43
signs of intoxication appear. A woman, who had taken 10 drops of mirbane oil
as an abortifacient, gave no indication of intoxication, that is to say, uncon-
sciousness and cyanosis, for 8 hours after taking the poison.
Nitrobenzene not only profoundly changes the blood but it irritates and
paralyzes the central nervous system (see R. Kobert, "Intoxikationen")-
Some nitrobenzene passes into the urine. Although it has been stated that
the organism does not convert nitrobenzene into aniline, Rossi 1 found in the
viscera of a person, who had died from supposed nitrobenzene poisoning,
aniline which evidently had been formed as a result of putrefaction. Conse-
quently in cases of fatal poisoning, tests for nitrobenzene should be made im-
mediately after death. In nitrobenzene poisoning human urine contains a
brown pigment but only rarely haemoglobin or methaemoglobin. Urine contain-
ing nitrobenzene will reduce Fehling's solution. It is also unfermentable and
distinctly laevo-rotatory. A conjugated glycuronic acid is possibly concerned
in these reactions.
Detection of Nitrobenzene
In nitrobenzene poisoning the urine and all the organs have
the odor of this compound. For the chemical tests the material
should first be distilled with water. Nitrobenzene distils quite
easily with steam and appears in the distillate as yellowish
globules. These are heavier than water and have a character-
istic odor. Vigorously agitate the globules, when separated as
completely as possible from water, with granulated tin and a
few cc. of concentrated hydrochloric acid, until there is no odor
of nitrobenzene. Pour the acid solution from undissolved tin,
and add an excess of potassium hydroxide solution to decompose
the double chloride of aniline and tin. Extract free aniline
with ether. Withdraw the aqueous liquid from the separating
funnel, and evaporate the ether extract spontaneously in a
small glass dish. Aniline, formed by reducing nitrobenzene,
will remain as globules which usually have a red or brown color.
Dissolve these globules by agitation with water, and use this
solution for the hypochlorite and phenylisocyanide tests (see
pages 45 and 36).
Mechanism of the Reaction. Nitrobenzene is reduced by nascent hydrogen
to aniline (a) which combines with the excess of hydrochloric acid forming aniline
hydrochloride (/3). From the latter compound potassium hydroxide liberates
aniline (y) :
1 Chemical Abstracts 9, 1341 (1915).
44 DETECTION OF POISONS
(a) C 6 H 6 -N0 2 + 6H = C 6 H 5 -NH 2 + 2 H 2 O,
03) C 6 H 6 -NH 2 + HC1 = C6H5-NH2.HC1 1 ,
M C 6 H 6 NH 2 .HC1 + KOH = C 6 H 5 -NH 2 + H 2 O + KC1.
ANILINE
Toxic Action. Aniline is moderately toxic in its action. Doses of 1.5 to 2
grams, administered in the course of a day, have proved fatal to small dogs. It
is not possible to state definitely the average lethal dose for human beings. Very
^r-rr serious results are said to have followed a dose of 3 or 4 grams of
aniline. The lethal dose is certainly less than 25 grams, for that
C quantity of aniline was sufficient to kill a healthy man. Even
^\ inhalation of aniline vapor may cause severe or fatal intoxications.
H 9 |T H Aniline produces methaemoglobin and therefore poisons the
HC CH blood. The conversion of oxyhaemoglobin into methaemoglobin
\/ by aniline may be demonstrated by adding an aqueous aniline
solution to blood in a test-tube. Aniline changes their form and
partially decomposes red blood-corpuscles. Thereby the quan-
tity of available oxygen in the blood is so diminished that it amounts to only 5
to 10 volumes instead of 15 to 20, the normal quantity. The number of red
blood-corpuscles is diminished in aniline poisoning but not that of the white
blood-cells.
R. v. Engelhardt has shown that aniline is partly changed in the human organ-
ism into aniline black, or into a similar compound insoluble in water. At the
climax of aniline poisoning blue-black granules may be seen in every drop of blood
and also in the urine. Aniline is oxidized in the system to para-aminophenol
(C6H4.OH,NH 2 (i,4)). Like all phenols this compound forms an ethereal sul-
phate with sulphuric acid, 2 namely, para-aminophenyl-sulphuric acid (HO.SO 2 .-
O.C 6 H 4 .NH 2 (i,4). This acid is eliminated through the kidnej^s as an alkali salt
and then appears in the urine. A part of the para-aminophenol is also elim-
inated as a conjugate of glycuronic acid. 3
The reduction of Fehling's solution by urine containing aniline is due to
this conjugated acid. In severe cases of poisoning unchanged aniline has also
been found in the urine. Usually urine that contains aniline has a very dark
color. Besides the substances mentioned, a dark pigment has been detected
1 Organic ammonium bases resemble ammonia in combining with acids to form
salts. Trivalent nitrogen of the free base is changed to pentavalent nitrogen in
the salt:
III /H
C 6 H 6 - N< + HC1 = C 6 H 6 - N~
\TT \~
\C1
Aniline Aniline hydrochloride
2 This conjugation takes place with elimination of water:
H 2 N.C 6 H 4 .OH + HO.S0 2 .OH = H 2 O + H 2 N.C 6 H 4 .O.SO 2 .OH (i, 4 )
3 Glycuronic acid, C 6 Hi O 7 = ^)C-(CH.OH) 4 -COOH, is closely related to
glucose. It is an uncrystallizable syrup. If its aqueous solution is boiled, the
acid is partly converted into the internal anhydride, glycurone (C 6 H S O 6 ), which
crystallizes well.
VOLATILE POISONS 45
in urine in aniline poisoning as well as haemoglobin, methaemoglobin and an
abundance of urobilin (R. Robert, "Intoxikationen").
Detection of Aniline
Aniline is a rather feebly base and part of it will pass over
with steam, when the acid solution is distilled. There will be
enough in the distillate for detection by the tests described
below. In estimating aniline quantitatively in any kind of mate-
rial the distillation must be as complete as possible. Mix the
substance with water, make strongly alkaline with sodium
hydroxide or carbonate solution and distil in a current of steam.
Since 30 parts of water at 15 dissolve i part of aniline, the
distillate may contain considerable of this amine. When the
quantity is large, oil-drops will appear. An aqueous aniline
solution (aniline water) colors pine wood and elder pith in-
tensely yellow. The following tests should be used for aniline :
1. Hypochlorite Test. Add a few drops of aqueous calcium
or sodium hypochlorite solution drop by drop to a portion of the
distillate. A violet-blue or purple- violet color, gradually chang-
ing to a dirty red, will appear if aniline is present. Addition of a
little dilute aqueous phenol solution containing some ammonia
will produce a blue color which is quite stable. This test is
sensitive i : 66,ooo. r
2. Phenylisocyanide Test. Heat a portion of the distillate
with a few drops of chloroform and potassium hydroxide solu-
tion. The repulsive odor of phenylisocyanide will show the
presence of aniline.
3. Bromine Water Test. Bromine water added to a solution
containing aniline will produce a flesh-colored precipitate. This
test is sensitive i : 66,000.
4. Chromic Acid Test. 2 Mix a trace of pure aniline with 4 to
5 drops of concentrated sulphuric acid in a porcelain dish and
add a drop of aqueous potassium dichromate solution. After
a few minutes the mixture beginning at the edge will take on a
1 Test this experimentally with very little aniline. For example, dissolve a
small drop in 30 cc. of water and take only 2-3 cc. of this dilute solution for the
test.
2 Beissenhirtz reaction, Annalen der Chemie und Pharmazie, 87, 376 (1853).
46 DETECTION OF POISONS
pure blue color. Addition of 1-2 drops of water produces at
once a deep blue color. To apply this test to the distillate,
first extract with ether, evaporate the ether solution and test an
oily residue as described.
CARBON DISULPHIDE
Carben disulphide, CS 2 , is a colorless liquid having a characteristic odor and a
high index of refraction. It is only slightly soluble in water. There is some
difference of opinion as regards the solubility of carbon disulphide in water.
1000 cc. of water dissolve
13-14 2 . 03 parts (Page)
15-16 i. 8 r. parts (Chancel; Par mentier)
15-16 2-3 ' parts (Ckindi)
15-16 3-S-4-S2 parts (Peligot)
Carbon disulphide is miscible in all proportions with absolute alcohol, ether,
ethereal and fatty oils.
Toxic Action. Carbon disulphide administered internally has a very poisonous
action upon the blood causing especially decomposition of red blood-corpuscles.
Even inhalation of carbon disulphide vapor frequently occasions severe poi-
soning. Carbon disulphide was formerly considered a typical producer of
methaemoglobin but recent investigations have not confirmed this opinion. It
has a very injurious action upon the red blood-corpuscles and causes haemolysis.
R. Robert (Intoxikationen) states that its power of dissolving lipoids is respon-
sible for its injurious action upon the blood and the central nervous system. E.
Harmsen 1 has recently come to practically the same conclusion. He considers
carbon disulphide a powerful blood poison because it decreases the number of red
blood-corpuscles and the quantity of haemoglobin and brings about a leuco-
cytosis. 2
Detection of Carbon Disulphide
Carbon disulphide distils very slowly with steam. Con-
sequently the second or third fraction of the distillate should
be used in testing for this substance. If 40 cc. are distilled
from 100 cc. of water containing 2 drops of carbon disulphide,
the following 10 cc. will give a distinct test. If the quantity
of carbon disulphide is small, it will remain in solution. Such
a solution does not have a strong odor. Carbon disulphide may
be recognized by the following tests :
1 Vierteljahrsschrift fur gerichtliche Medizin, 30, 442 (1905).
2 Leucocytosis means a temporary increase in the number of white blood-
corpuscles (leucocytes) as compared with the number of red blood-corpuscles.
Normally there are about 350 red to i white blood-corpuscle, whereas in leucocy-
tosis the proportion is 20 : i.
VOLATILE POISONS 47
1. Lead Acetate Test. Add a few drops of lead acetate
solution to the liquid containing carbon disulphide. It will
cause neither a precipitate (distinction between 82 and H^S)
nor a color. Add excess of potassium hydroxide solution and
boil. A black precipitate (PbS) will appear. This is a very
delicate test.
2. Sulphocyanate Test. Heat an aqueous solution of carbon
disulphide for a few minutes with concentrated ammonium
hydroxide solution and alcohol. Ammonium sulphocyanate
(H 4 NSCN) is formed together with ammonium sulphide.
Concentrate this solution upon the water-bath to about i cc.
and acidify with dilute hydrochloric acid. Add a drop of
ferric chloride solution and a deep red color will appear. This
test will show even traces of carbon disulphide, for example
0.05 gram in i cc. of water.
Mechanism of the Reaction :
(a) 4NH 3 + CS 2 . = (H 4 N)SCN + (H 4 N) 2 S,
(0) FeCl 3 + 3 (H 4 N)SCN = Fe(SCN) 3 + 3(H 4 N)C1.
3. Xanthogenate Test. Shake a few cc. of distillate for
several minutes with 3 or 4 times its volume of saturated solu-
tion of potassium hydroxide in absolute alcohol. Faintly
acidify the solution with acetic acid and add i or 2 drops of
copper sulphate solution. If carbon disulphide is present, a
brownish black precipitate of cupric xanthogenate will appear.
This will soon change to a yellow, flocculent precipitate of
cuprous xanthogenate, CS(SCu) (OC 2 H 5 ). Vitali's procedure is
somewhat different and consists in adding solid ammonium
molybdate to the alkaline reaction-product and then in acidify-
ing with dilute sulphuric acid. The appearance of a red color
indicates carbon disulphide.
Mechanism of the Reaction. Alcoholic potassium hydroxide acts like potas-
sium alcoholate (C 2 H 5 -OK) and converts carbon disulphide into potassium
xanthogenate
/SK
CS 2 + C 2 H 5 -OK = C=S
\OC 2 H 5
This compound treated with cupric salts gives first a brownish black precipitate
of cupric xanthogenate:
/SK ' /S
2C==S + CuS0 4 = (S = C( ) 2 Cu + K 2 SO 4
\OC 2 H 5 X OC 2 H 5
4g DETECTION OF POISONS
The cupric salt then forms cuprous xanthogenate andethyl xanthogen disulphide:
/OC 2 H 6 /OC 2 H 5
s = c s = c< s = c\
X)C 2 H B X OC 2 H 6 X OC 2 H 6
Cupric Ethyl xanthogen Cuprous
xanthogenate disulphide xanthogenate
Quantitative Estimation of Carbon Disulphide in Air
Inhalation of air containing carbon disulphide has frequently given rise to
chronic poisoning. Persons thus affected have usually been laborers in rubber fac-
tories. Consequently experiments have been made to determine the maximum
quantity of carbon disulphide air may contain without injury to health. The
results of these investigations may be summarized as follows:
CS 2 in mgrs.
per liter of air
1. 0.5-0.8 No injurious effect.
2. 1.3 Slight uneasiness after several hours.
3. - 3.4 Uneasiness in 30 minutes.
4. 6.0 Uneasiness in 20 minutes.
5. 10.0 Paralysis attended by after-effects last-
ing several days.
The exact danger limit for persons obliged to live for weeks at a time in an atmo-
sphere containing carbon disulphide should be placed below 3 mg. per liter
of air. Air in factories, where operatives work in presence of carbon disulphide
vapor, should never exceed this limit. In rubber factories the air is said fre-
quently to contain 2.5 to 3 mg. per liter. Since experiments have shown that
93 to 96 per cent, of the carbon disulphide breathed was exhaled unchanged, an
exceedingly small quantity is capable of producing toxic symptoms.
Procedure. Place a saturated alcoholic solution of potassium hydroxide in a
Peligot absorption-tube and draw through this solution 10 to 20 liters of air con-
taining carbon disulphide vapor. A quantitative formation of potassium xan-
thogenate (see above) will take place.
Dilute the contents of the receiver at the. end of the experiment with 96 per
cent, alcohol and bring the volume to 50 cc. Measure an aliquot portion of this
solution and dilute with water. Faintly acidify the solution with acetic acid
and remove excess- of acid with acid sodium carbonate. Add freshly prepared
starch solution and o.i n-iodine solution until there is a permanent blue color.
Iodine converts potassium xanthogenate according to equation (I) into ethyl
xanthogen-disulphide :
KS.CS.OC 2 H 6 S.CS.OC 2 H 5
I. Ii + = 2KI + |
KS.CS.OC 2 H 2 S.CS.OC 2 H 5
VOLATILE POISONS 49
E. Rupp and L. Krauss 1 think the action of iodine upon potassium xanthogen-
ate is expressed by equation (II) :
II. 2 KS.CS.OC 2 H 6 + H 2 + 2! = KS.CS.SK + 2 C Z H 5 .OH + aHI + S.
Both equations require the same quantity of iodine, namely, 2 atoms for 2
molecules of xanthogenate. A difference therefore in the mechanism of the
reaction has no influence on the combining relations of the iodine and the method
is applicable to the quantitative determination of xanthogenate.
1000 cc. of o.i n-iodine solution, containing o.i gram-atom of iodine, corre-
spond to o.i gram-molecule of CSz = 7.6 grams.
ETHYL ALCOHOL 2
Fate in the Human Organism. Ethyl alcohol brought in contact with many
different parts of the organism is very rapidly absorbed, but especially easily
from an empty stomach. Although there is practically no absorption of non-
volatile aqueous liquids from the stomach, ethyl alcohol, is freely
absorbed. After absorption it passes into the blood and is then
jj C H distributed to all organs (see chloral hydrate). Experiments upon
dogs, colts and adult horses (see Kobert, " Intoxikationen ") have
^ shown that blood at the climax of narcosis contains 0.72 per cent,
jj of ethyl alcohol . There is stupor even when o. 1 2 per cent, is present.
There is difference of opinion among toxicologists regarding
alcoholic intoxication, as to whether the poison is distributed uniformly through-
out the body, or accumulated in the brain in larger quantity than in other
organs. The following percentages of ethyl alcohol, found in the organs of a
man, who had died at the climax of severe acute ethyl alcohol poisoning, lend
support to the latter view: liver 0.21, brain 0.47 and blood 0.33 per cent. It
appears from these results that the brain takes up an especially large quantity of
ethyl alcohol.
Uncertainty concerning the subsequent fate of ethyl alcohol in the organism
has finally been removed. Experiments have shown that ethyl alcohol is never
eliminated unchanged through the skin. At most only 1-1.5 per cent, passes
off through the kidneys and only 1.6-2 per cent, through the lungs. Strass-
mann 3 found the quantity eliminated by the lungs somewhat higher (5-6 per
cent.) and by the kidneys 1-2.5 P er cent. The remainder is completely oxidized
in the human organism to carbon dioxide and water.
B. Fischer found the following quantities of ethyl alcohol in organs removed
from a man who had probably died from drinking too much brandy:
Weight Organ Ethyl Alcohol
2720 grams Stomach and intestines 30.6 grams
2070 grams Heart, lungs and blood 10.85 grams
1820 grams Kidneys and liver 7.8 grams
1365 grams Brain 4.8 grams
1 Berichte der Deutschen chemischen Gesellschaft 35, 4257 (1902).
2 The word "alcohol," unqualified by an adjective, i.e., methyl, amyl, etc.,
means ethyl alcohol. Tr.
3 Pfliiger's Archiv, 49, 315 (1891).
50
DETECTION OF POISONS
Detection of Ethyl Alcohol
Ethyl alcohol distils easily with steam and consequently
most of it will be in the first fraction. If present in sufficient
quantity, it can be recognized in the distillate by its odor. The
following tests should be made:
1. Lieben's lodoform Test. 1 Gently warm the liquid (40-
50), add a few cc. of aqueous iodo-potassium iodide solution, or
a small crystal of iodine, and enough potassium hydroxide
solution to give the liquid a distinct
yellow to brownish color. If, ethyl
alcohol is present, a yellowish white
to lemon-yellow precipitate of iodo-
form will appear immediately, or as
the solution cools. If the quantity
of ethyl alcohol is very small, a
precipitate will form on long stand-
ing. When iodoform is deposited
slowly, the crystals are very perfect
PIG. 9 .-Iodof orm Crystals. hexagonal plates and sta rs (see Fig. 9) .
Note. This iodoform test is very delicate but not characteristic of ethyl
alcohol, since other primary alcohols, except methyl alcohol, and many secondary
alcohols, as well as their oxidation products, aldehydes and ketones, give iodo-
form under the same conditions. Acetic ether, aceto-acetic ether, lactic acid,
etc., also give iodoform.
The correct explanation of the iodoform reaction is probably the following:
Iodine and potassium hydroxide form potassium hypo-iodite (KOI) by reaction
(a). This compound brings about the oxidation of ethyl alcohol to acetic
aldehyde (/3) and at the same time substitutes iodine for hydrogen in the latter
(y). Finally tri-iodo-acetic aldehyde is decomposed by the excess of potassium
hydroxide into iodoform and potassium formate (6) :
(a) 2KOH + I, = KI + H 2 O + KOI,
. (/3) CH 3 .CH 2 .OH + KOI = CH 3 .CHO + H 2 O + KI,
(7) CH 3 .CHO + 3KOI = 3 KOH + CI 3 .CHO,
(5) CI 3 .CHO + KOH = CHI 3 + H.COOK.
2. Berthelot's Test. Shake the liquid containing ethyl
alcohol with a few drops of benzoyl chloride and about 5 cc. of
sodium hydroxide solution (10 per cent.), until the irritating
1 Annalen der Chemie und Pharmazie, Supplement Band, 7, 218.
VOLATILE POISONS 51
odor of benzoyl chloride has gone. The aromatic odor of
ethyl benzoate will appear.
C 6 H 6 .COC1 + C 2 H 5 .OH + KOH = C 6 H 5 .CO.OC 2 H 5 + KC1 + H 2 O
Ten cc. of 0.5 per cent, ethyl alcohol will give a distinct odor
of this ester.
3. Chromic Acid Test. Warm the liquid containing ethyl
alcohol with dilute sulphuric, or hydrochloric acid, and add i
or 2 drops of very dilute potassium dichromate solution. The
color of the liquid will change from red to green, and at the same
time the odor of acetaldehyde will be recognized. This test is
not characteristic of ethyl alcohol because many other volatile
organic compounds behave similarly.
Mechanism of the Reaction
(a) K 2 Cr 2 O 7 + H 2 S0 4 = K 2 SO 4 + H 2 Cr 2 O7(H 2 O + sCrO,),
(0) 3 C 2 H 6 .OH + 2 Cr0 3 + 3H 2 S0 4 = 3 CH 3 .CHO + Cr 2 (SO 4 ) 3 + 6H 2 O.
% Acetaldehyde
4. Ethyl Acetate Test. Mix the liquid containing ethyl
alcohol with the same volume of concentrated sulphuric acid.
Add a very small quantity of anhydrous sodium acetate and
heat. Ethyl acetate will be recognized by its odor.
() C 2 H 6 .OH + H 2 SO 4 = CjHsO.SOa.OH 1 + H 2 O,
(/3) CH 3 .CO.ONa + C 2 H 6 O.SO 2 .OH = CH 3 .CO.OC 2 H 5 + NaHSO 4 .
5. Vitali's Test. Thoroughly mix a few cc. of distillate in a
glass dish with a small piece of solid potassium hydroxide and
2 or 3 drops of carbon disulphide. Let this mixture stand for a
short time without warming. When most of the carbon disul-
phide has evaporated, add a drop of ammonium molybdate
solution and then an excess of dilute sulphuric acid. If the dis-
tillate contains ethyl alcohol, a red color will appear. Potas-
sium xanthogenate (CS(OC 2 H 5 )(SK)) is first formed. This
compound gives a red color with ammonium molybdate.
Acetone and acetaldehyde produce a similar color. This test is
given distinctly by 5 per cent, aqueous ethyl alcohol solution.
<X OC 2 H 5
1 The structural formula of ethyl sulphuric acid is
52 DETECTION OF POISONS
METHYL ALCOHOL
TT Woods of various kinds, but more especially the so-called hard
woods such as maple, beech, birch, etc., when subjected to dry dis-
H C H tillation, yield a complex mixture of many substances among which
is wood spirit. The latter carefully freed of attendant impurities
by chemical means and distillation is known as wood or methyl
alcohol.
Pure methyl alcohol is a colorless liquid having an agreeable alcoholic odor
and boiling at about 66. It is miscible with water in all proportions. Com-
bined chemically with other substances, it occurs in nature; for example, as
methyl salicylate in oil of wintergreen. Methyl alcohol has many industrial
applications. It is extensively used as a solvent and in making other com-
pounds. The crude alcohol in considerable quantity furnishes a means of
denaturing grain alcohol, since its disagreeable odor renders the latter obnoxious
as a beverage but does not unfit it for many manufacturing processes. Methyl
alcohol is also frequently employed as an adulterant of ethyl alcohol and as a
substitute for the latter, especially in tinctures and varnishes. These uses,
however, are not legitimate and should be condemned.
Toxic Action. Methyl alcohol is a subtle and dangerous poison. Since it
is used not only in adulterating spirituous liquors but in preparing Jamaica
ginger, peppermint and lemon extracts, cologne, bay rum, Florida water and
witch hazel, as well as lacquers and varnishes, its deleterious effects are to be
expected whenever such preparations are taken internally. The increasing diffi-
culty of obtaining in the United States beverages that contain ethyl alcohol has
led unscrupulous and ignorant persons to substitute in its place the more acces-
sible but more deadly methyl alcohol.
In the case of animals a single dose of methyl alcohol has been found less toxic
than the same quantity of ethyl alcohol. Also its action is slower but of longer
duration. More marked gastric irritation and convulsive movements frequently
have been observed. If administered repeatedly, methyl alcohol is much more
toxic than ethyl alcohol. Von Pohl 1 has shown that methyl alcohol unlike
ethyl alcohol is incompletely oxidized in the organism. In consequence formic
acid and possibly formaldehyde, both more toxic than methyl alcohol, are formed
in the tissues.
In man methyl alcohol gives rise to marked muscular weakness and defective
heart action. Nausea, vomiting, coma and a delirium that is much more in-
tense and persistent than that attending intoxication by ethyl alcohol subse-
quently have been observed. Methyl alcohol frequently has proved lethal,
which would not have been the case had the same quantity of ethyl alcohol been
taken. Cases are on record where the victims have recovered only to be totally
and permanently blinded because of optic neuritis and subsequent complete
optic atrophy. There can be no doubt therefore that methyl alcohol is a very
dangerous poison which should never be administered internally nor applied
externally in a manner making possible its entrance into the body through
any of the ordinary channels of absorption.
1 Therapeutische Monatshefte 1892, page 327.
VOLATILE POISONS 53
Detection of Methyl Alcohol
Methyl alcohol distils easily with steam and will appear
mainly in the first fraction. When pure it may be recognized
by its boiling-point but usually it is necessary to detect a small
quantity in presence of vegetable or animal extractive matter
and of other volatile liquids or solids.
Isolation of Methyl Alcohol from Mixtures. It is not advisable to undertake
the detection or estimation of methyl alcohol in mixtures containing other
volatile products unless a preliminary purification has been made. Thorpe
and Holmes 1 bring the volume of 25 cc. of the liquid to 100-150 cc. with water
in a separatory funnel, saturate with sodium chloride and shake well for 5
minutes with 50-80 cc. of petroleum ether boiling below 60. Withdraw the
under layer after 30 minutes and, if necessary, extract again. Shake the pe-
troleum ether extracts successively a second time with 25 cc. of saturated salt
solution. Mix the original solution with the brine washing in a distilling flask,
neutralize any acidity with sodium hydroxide solution and collect 25-50 cc.
of distillate. Bring the volume of the distillate to 100 cc. and use this solution
for tests.
Several tests depend upon first oxidizing methyl alcohol to
formaldehyde and then detecting the latter by means of a color
change.
1. Copper Oxidation Test. 2 Dilute the solution, if neces-
sary, until the total alcoholic strength does not exceed 10 per
cent. Immerse in cold water the test-tube containing 3 cc. of
liquid and insert 3-4 times a copper spiral heated to redness,
reheating each time. Filter, expel by boiling any odor of acetic
aldehyde, cool and add one drop of 0.5 per cent, aqueous re-
sorcinol solution. Add this mixture from a pipette^ to form an
upper layer on 2 cc. of concentrated sulphuric acid and gently
rotate the test-tube for 3 minutes. If a rose-red ring does not
appear at the contact-surface of the two liquids, less than 2 per
cent, of methyl alcohol is present.
2 . Permanganate Oxidation Test. The following methods
have been proposed:
(a) Scudder-Biggs 3 Method. Add 0.5 cc. of concentrated
1 Journal of the Chemical Society 83, 314 (1903).
2 Mulliken and Scudder: American Chemical Journal 21, 266 (1899).
3 Journal of the American Chemical Society 28, 1202 (1906).
54 DETECTION OF POISONS
sulphuric acid and 5 cc. of saturated potassium permanganate
solution to 10 cc. of liquid, keeping the temperature at 20-25.
After 2 minutes discharge the color by means of sulphurous acid,
expelling the latter and any acetic aldehyde by heat. Complete
the test with resorcinol and sulphuric acid as in the copper wire
test. In presence of ethyl alcohol, less acetic aldehyde is
said to be produced by permanganate than by copper.
(b) United States Pharmacopoeia 1 Method. This method
is employed to test for methyl alcohol in ethyl alcohol. The
liquid should contain not more than 10 per cent, of ethyl alco-
hol by volume. Add 2 cc. of potassium permanganate solu-
tion (3 grams of KMnO 4 in 100 cc. of distilled water) and 0.3 cc.
of sulphuric acid to 5 cc. of the liquid. Dissolve the precipi-
tated manganese dioxide after 5 minutes by adding sulphurous
acid drop by drop and shaking. Then add i cc. of sulphuric
acid, 5 cc. of fuchsin-sulphurous acid solution, 2 and mix thor-
oughly. If methyl alcohol is present, the liquid will be color-
less after 10 minutes standing.
(c) Deniges 3 Simmonds 4 Method. The total alcoholic
strength of the liquid should not exceed 10 per cent, by volume.
Add to 5 cc. of the liquid 2.5 cc. of potassium permanganate
solution (2 per cent.) and 0.2 cc. of concentrated sulphuric acid.
After 3 minutes follow with 0.5 cc. of oxalic acid solution (9.6
grams of crystals in 100 cc.). When shaken, the mixture be-
comes clear and nearly colorless. Then run in i cc. of concen-
trated sulphuric acid, mix well and finally add 5 cc. of Scriiff's
reagent. 5 In a few minutes a violet color will appear, if more
than traces of methyl alcohol are present, otherwise it may take
20-30 minutes.
3. Hinkel's Test. 6 Add to i cc. of liquid 0.8 gram of am-
1 United States Pharmacopoeia, Ninth Decennial Revision, page 36.
2 Add 10 cc. of hydrochloric acid to a solution of 0.5 gram of fuchsin and 9
grams of sodium bisulphite in 500 cc. of distilled water.
4 Comptes rendus de 1' Academic des Sciences 150, 832 (1910).
4 Analyst 37, 16 (i9T2>.
Dissolve 0.2 gram of rosaniline base in 20 cc. of cold saturated sulphurous
acid solution. If the color is not discharged in 24 hours, add 10 cc. more of sul-
phurous acid. Repeat, if necessary, until there is no color and dilute to 200 cc.
6 Analyst 33, 417 (1908).
VOLATILE POISONS 55
monium persulphate (H 4 N.SO 4 ) and 3 cc. of dilute sulphuric
acid d : 5). Dilute the mixture to 20 cc. with water and distil,
collecting in test-tubes 5 separate 2 cc. portions. Reject the
first two portions, which contain any acetic aldehyde, and add
to the last three a few drops of 0.5 per cent, morphine hydro-
chloride solution. Finally run into each test-tube 3 cc. of
concentrated sulphuric acid as an under layer. A violet ring
will appear at the contact-surface of the two liquids, if formal-
dehyde is present. This test will not show with certainty less
than 5 per cent, of methyl alcohol in ethyl alcohol.
Quantitative Estimation of Methyl Alcohol
The method of Leach and Lythgoe, 1 requiring the use of the
Zeiss immersion refractometer, furnishes a rapid means of
estimating methyl alcohol in presence of ethyl alcohol. This
instrument gives a reading of 14.5 for distilled water at 20, a
maximum reading of 101 for 75 per cent, ethyl alcohol and 91
for 100 per cent.; and a maximum reading of 39.8 for 50 per
cent, methyl alcohol, 14.9 for 91 per cent, and 2 for 100 per
cent. 2
ACETONE
Human urine almost always contains a very small quantity of acetone as a
physiological constituent. Under pathological conditions, especially in diabetes
jj mellitus (diabetic acetonuria), urine contains much more. It is
also present in urine in protracted high fever, digestive disturb-
H C H ances, severe forms of carcinoma (carcinomatous acetonuria), etc.
I Finally, acetone has been found in urine in considerable quantity
| ~ in various intoxications (toxic acetonuria), for example, in poison-
H C H ing by phosphorus, carbon monoxide, atropine, curare, antipyrine,
pyrodine, sulphuric acid, extract of male fern; in chronic lead
poisoning; and in chronic morphinism after discontinuance of the
drug (see R. Robert, "Intoxikationen").
Acetone is not poisonous nor in the least corrosive. Man and animals can
tolerate considerable quantities of acetone taken internally. It seems to produce
no effect, though it may possibly possess very feeble narcotic properties. Arch-
angelsky found dogs to show signs of narcosis when the blood contained 0.5 per
cent, of acetone. Even smaller doses produce narcosis in rabbits and have an
injurious action upon the blood and kidneys.
1 Journal of the American Chemical Society 27, 964 (1905).
2 Tables giving the percentages of methyl alcohol corresponding to the dif-
ferent readings will be found in the original communication of the authors.
56 DETECTION OF POISONS
Distillates from human urine, as well as from blood and various organs, as
liver, spleen, kidneys, brain, etc., often contain acetone, or more correctly per-
haps, substances like acetone. This is especially the case when cadaveric mate-
rial has begun to putrefy.
Acetone is a clear, colorless liquid boiling at 56. It has a peculiar, fruity odor
and is neutral in reaction. It is miscible in all proportions with water, ethyl
alcohol and ether. It distils easily with steam.
Detection of Acetone
i. Lieben's lodoform Test. Add a few cc. of aqueous iodo-
potassium iodide solution, or a small crystal of iodine, to an
aqueous solution of acetone and then potassium hydroxide
solution drop by drop until the color is yellow. lodoform
immediately separates, even in the cold, as a yellowish white
precipitate which is usually amorphous. Acetone differs from
ethyl alcohol in giving iodoform, when ammonium hydroxide
solution is substituted for potassium or sodium hydroxide
solution (Gunning's acetone test).
Acetaldehyde resembles acetone in giving iodoform in the
cold and under conditions the same as those stated above.
Note. Potassium hypo-iodite (a) probably converts acetone into tri-iodo-
acetone (CHj.CO.CIs) (/3) and this compound is then decomposed by potassium
hydroxide into iodoform and potassium acetate (y) :
(a) 6KOH + 3 I 2 = 3 KI + 3 KOI
(0) CH,.CO.CH 3 + 3 KOI = CH 3 .CO.CI 3
(7) CHs.CO.CIs 4- KOH = CHI 3 + CH 3 .CO.OK.
2. Legal's Test. Add a few drops of freshly prepared sodium
nitroprusside solution to a liquid containing acetone, and then
potassium hydroxide solution. A red or reddish yellow color
will appear. This color soon changes to yellow. Add an
excess of acetic acid to the solution. The solution will now
have a carmine to purplish red color, according to the quantity
of acetone present. Heat will change this color to violet.
Ethyl alcohol does not give Legal's test, though acetaldehyde does. The red
color caused by aldehyde fades upon addition of acetic acid, and changes to green
with heat. Le Nobel states that ammonium hydroxide, or ammonium carbonate
solution, may be substituted for potassium hydroxide solution in Legal's test,
but under these conditions the red color is very slow to appear. Le Nobel's
modification, however, eliminates the possibility of confusing acetone with acet-
aldehyde.
VOLATILE POISONS 57
3. Penzoldt's Test. Prepare a hot, saturated, aqueous
ortho-nitro-benzaldehyde (C 6 H 4 .NO 2 .CHO(i,2)) solution and
allow it to cool. Add this solution to the liquid containing
acetone, and also some sodium hydroxide solution. At first
the color of the mixture is yellow. It then becomes green, and a
blue precipitate of indigotin is formed in 10 to 15 minutes.
When indigotin is present in traces only, shake the solution with
chloroform. This solvent will dissolve the coloring matter and
become blue.
4. Reynolds' Test. Acetone will dissolve freshly precipi-
tated mercuric oxide, and this test is based upon this property.
Add mercuric chloride solution to the distillate, and an alcoholic
potassium hydroxide solution. Shake thoroughly and filter.
Add ammonium sulphide solution to the clear filtrate as an upper
layer. If acetone is present, there will be a black zone (HgS)
where the two solutions meet.
Detection of Acetone in Urine. Acidify 200 to 500 cc. of urine with a few drops
of sulphuric acid and distil. Collect 20 to 30 cc. of distillate. This will contain
the entire quantity of acetone in the urine. Acetone thus obtained may pos-
sibly be derived from aceto-acetic acid which is often present in human urine,
especially in a severe case of diabetes mellitus. Distillation decomposes this
acid into acetone and carbon dioxide.
CH 3 .CO.CH 2 .CO.OH = CH 3 .CO.CH 3 + CO 2 .
Detection of Ethyl Alcohol and Acetone in Mixtures. Ethyl alcohol may be
detected in presence of acetone by Berthelot's test. On the other hand, ace-
tone may be distinguished from ethyl alcohol by Legal's or Penzoldt's test.
BITTER ALMOND WATER AND BENZALDEHYDE
Bitter almond water (Aqua Amygdalee Amarae of the Phar-
macopoeia) contains hydrocyanic acid. Only a small portion
of this acid, however, is free so that it can be precipitated by
silver nitrate solution. The greater part is combined as the
/ H
cyanohydrin of benzaldehyde, C 6 H 5 .C^OH, which does not re-
act with silver nitrate. But potassium hydroxide solution
will decompose this compound.
C 6 H 5 CH(OH)CN + KOH = KCN + H 2 O + C 6 H 5 .CHO.
58 DETECTION OF POISONS
Pure benzaldehyde, also called hydrocyanic acid-free oil of
bitter almonds, is not poisonous. It is oxidized to benzoic
acid in the body and eliminated in the urine partly as that acid
and. partly as hippuric acid after conjugation with glycocoll
(amino-acetic acid) :
(a) C 6 H 5 -CHO + O = C 6 H 6 -COOH,
(/3) CeHs-COOH + H 2 N-CH 2 -COOH = C 6 H 6 -CO-NH-CH 2 -COOH.
Benzoic acid Glycocoll furnished Hippuric acid
by the organism
Ordinary commercial oil of bitter almonds contains hydro-
cyanic acid and is poisonous in proportion to the quantity of
this acid present.
Test for hydrocyanic acid by shaking about 2 cc. of oil of
bitter almonds with 20 cc. of potassium hydroxide solution and
making the Prussian blue test. When oil of bitter almonds is
mixed with other material, distil with steam from a solution
acidified with tartaric, or dilute sulphuric acid, and test the first
part of the distillate for hydrocyanic acid. If benzaldehyde is
present, the distillate at the same time will be milky and have
the characteristic odor of that compound. Distil until the drops
of water are perfectly clear. Benzaldehyde may be detected
with certainty, and at the same time distinguished from nitro-
benzene which has a somewhat similar odor, by adding a few
drops of potassium hydroxide solution to the milky distillate,
to combine with any hydrocyanic acid, and extracting with
ether. The ether upon evaporation will deposit benzaldehyde
as globules, which can be positively identified by conversion
into benzoic acid. Heat the globules for a few minutes in a
small flask, attached to a reflux condenser, with about 10 cc. of
potassium dichromate solution and a little dilute sulphuric acid.
Cool, extract with ether and evaporate the ether solution in a
glass dish. When the material contains benzaldehyde, this
residue will consist of benzoic acid. This substance may be
further identified by its melting-point (120-121), its property
of subliming and the test with ferric chloride solution. 1
1 Dissolve the residue in a small quantity of water, and neutralize benzoic acid
by heating the solution to boiling with excess of calcium carbonate. Filter and
add a few drops of ferric chloride solution. If benzoic acid is present, a flesh-
colored precipitate of basic ferric benzoate will appear. Tr.
VOLATILE POISONS 59
SYNOPSIS OF GROUP I
Scherer's Test for Phosphorus Precedes Distillation
The material to be examined must, first be rendered uniform
by grinding or chopping. Add sufficient water to thin the mass,
acidify with tartaric acid and distil. If the preliminary test
for phosphorus (Scherer's) is positive, distil in the Mitscherlich
apparatus; otherwise distil as usual with a Liebig condenser.
It is advisable to collect the distillate in two or three fractions.
Test the first 5 to 10 cc. of distillate for hydrocyanic acid,
chloroform, ethyl and methyl alcohol, acetone and possibly
also for nitre-benzene and iodoform. Use the remainder of
the distillate in testing for carbolic acid, chloral hydrate and
carbon disulphide.
Phosphorus. Phosphorescence in Mitscherlich apparatus
during distillation in a dark room. Evaporate distillate with
strong chlorine water, or a little fuming nitric acid, and test the
residue for phosphoric acid. As an alternative procedure,
examine the original material, or at least the Mitscherlich dis-
tillate, for phosphorus by the Blondlot-Dusart method.
Hydrocyanic Acid. Odor. Schonbein's preliminary test.
Prussian blue test. Sulphocyanate test. Nitroprusside test.
Silver nitrate test. Alkaline phenolphthalin test.
Carbolic Acid. Odor. Red color with MilloH's reagent.
Yellowish white precipitate with bromine water. Violet color
with ferric chloride solution.
Chloroform. Separation of colorless globules, when the
quantity is large. Odor. Phenylisocyanide test, when heated
with aniline and potassium hydroxide solution. Reduces
silver nitrate and Fehling's solutions with heat. Red color
with resorcinol and potassium hydroxide solution. Blue color
with naphthol and potassium hydroxide solution. Blue-red
color with pyridine and sodium hydroxide solution.
Chloral Hydrate. Gives chloroform reactions. Brick-red
precipitate with Nessler's solution which in time becomes
yellowish green. Gives chloroform and magnesium formate,
60 DETECTION OF POISONS
when heated with magnesium oxide and water. Test for
formate with silver nitrate or mercuric chloride solution.
lodoform. Odor. Distillate milky and yellowish white.
Ether extract of distillate leaves crystals upon evaporation,
gives chloroform reactions.
Nitrobenzene. Yellowish globules with characteristic odor.
Reduced to aniline, when shaken with tin and hydrochloric
acid. Test for aniline.
Aniline. Violet color with, calcium hypochlorite solution.
Phenylisocyanide test, when heated with chloroform and
potassium hydroxide solution. Flesh-colored precipitate with
bromine water. Dark red color on warming with Millon's
reagent.
Carbon Bisulphide. Black precipitate, or only black colora-
tion (PbS), when heated with lead acetate and potassium
hydroxide solutions. Formation of ammonium sulphocyanate
by evaporation with concentrated ammonium hydroxide solu-
tion and detection with ferric chloride solution. Formation
of potassium xanthogenate, when shaken with alcoholic solu-
tion of potassium hydroxide and detection with copper sulphate
solution.
Ethyl Alcohol. lodoform test. Odor of ethyl benzoate,
when shaken with benzoyl chloride and sodium hydroxide solu-
tion. Green color and aldehyde odor, when heated with potas-
sium dichromate and hydrochloric acid- Vitali's test.
Methyl Alcohol. Oxidation tests with copper, potassium
permanganate and ammonium persulphate.
Acetone. Gives iodoform, even in the cold, with iodine
and potassium hydroxide or ammonium hydroxide solution.
Legal's test. Indigotin test. Reynolds' test.
CHAPTER II
NON-VOLATILE POISONS 1
Alkaloids, Glucosides and Synthetic Compounds Non-volatile with Steam
from Acid Solution
Put a portion of finely chopped material into a large flask,
and thoroughly mix with two or three times as much absolute
ethyl alcohol. 2 Add enough tartaric acid solution to give the
mixture a distinct acid reaction after shaking. Laboratory
experiments usually require 20 to 30 drops of 10 per cent, tartaric
acid solution. Avoid a large excess of tartaric acid, since it
may act as an objectionable impurity in the ether extract, owing
to its solubility in that solvent. Connect the flask with a glass
tube (80 to 100 cm. long) serving as a reflux cooler. Frequently
shake and heat 10 to 15 minutes upon the water-bath. In the
exti action of a large quantity of material from a cadaver, con-
nect the flask with an upright Liebig condenser used as a reflux
cooler (Fig. -10). Cool the flask contents and filter to remove
fat and other insoluble matter. Wash the residue with ethyl
alcohol. Evaporate the filtrate, which must have an acid reac-
tion, to a thin syrup in a glass dish upon the water-bath. Thor-
oughly mix this residue with 100 cc. of cold water. Usually
this causes an abundant separation of fat and resinous matter,
especially when parts of a cadaver are examined. Filter and
evaporate the nitrate to dryness, or to a syrup, upon the water-
1 The isolation of these toxic substances from cadaveric material, food, etc.,
is necessary before tests establishing their presence can be made. Mixtures used
for laboratory practice, consisting of dog biscuit, meat, comminuted organs (liver,
kidneys, spleen), sausage meat, etc., with any of the poisons of this group, should
be examined according to the method outlined above.
2 Commercial ethyl alcohol usually contains basic compounds, the presence
of which is objectionable. They should be removed by adding tartaric acid to
the ethyl alcohol and distilling. Ethyl alcohol should not be used in toxicologi-
cal analysis, unless an actual test has shown it to be free from such impurities.
Tr.
61
62
DETECTION OF POISONS
c
bath. Thoroughly mix this residue with absolute ethyl alcohol.
As a result of this treatment, a whitish substance, which is more
or less viscous or slimy, usually remains undissolved. This
residue, which consists chiefly of protein substances (albumin,
albumoses and peptones), dextrin-like compounds and in part
also of inorganic salts, frequently becomes
granular upon standing. Tartrates of the
alkaloids and other organic poisons are
dissolved. The larger the quantity of ab-
solute ethyl alcohol used, the more com-
plete the precipitation of those substances
., which interfere more or less with the
t^^r** ^" detection of organic poisons. Again evap-
orate the filtered alcoholic solution upon
the water-bath, and dissolve the residue
in about 50 cc. of water. If the solution
is not perfectly clear, filter through a
moistened paper.
The result of this procedure is a solution
containing alkaloidal tartrates and other
organic substances belonging to this
group. This solution should have an
acid reaction and be practically free from
protein substances, fat, resinous bodies
and coloring matter. If the solution
fulfils these requirements, it is ready to
be examined for organic poisons accord-
ing to the "Stas-Otto" method. The
utmost care must be taken in preparing
this solution, because definite conclusions
cannot be drawn from the uncertain tests given by impure
material.
For the purpose of isolating alkaloids from viscera free from
ptomaines and other impurities, Magnin and Zappi 1 suggest
the following procedure. Finely comminute the material and
macerate in water acidulated with a few drops of dilute sul-
1 Chemical Abstracts 9, 2748 (1915).
NON-VOLATILE POISONS 63
phuric acid (i :4). Warm at 40 for 1-2 hours to facilitate
extraction and set aside for 18-20 hours. Filter, add an equal
volume of 90-92 per cent, ethyl alcohol to the filtrate, then
10 cc. of 30 per cent, aluminium sulphate solution and finally
10 cc. of 15 per cent, potassium hydroxide solution. After
2-3 hours filter and concentrate the filtrate in vacuo to a sirupy
consistency at a temperature not exceeding 45-50. Treat
the residue with 20 times its volume of 90-92 per cent, ethyl
alcohol, set aside for 18-20 hours, filter and again concentrate
in vacuo as above. Dissolve the residue in water and use the
solution for the extraction of alkaloids. Ptomaines and other
impurities are almost completely eliminated by this procedure.
When the material is a powder mixed with cane- or milk-
sugar, it is usually possible, after the aqueous solution has been
acidified with tartaric acid, to extract diiectly with ether and
continue according to the Stas-Otto method.
Frequently in suspected poisoning an examination of beer,
wine, black coffee, infusion of tea, food, etc., is necessary. In
such cases the process outlined above may often be considerably
shortened. Acidify the material with aqueous tartaric acid
solution, if necessary, and evaporate in a glass dish upon the
water-bath. Treat the residue thoroughly with absolute ethyl
alcohol and filter. Evaporate the filtrate uporr the water-
bath and dissolve the residue in tepid water. Filter this
solution, if necessary, and then examine according to the
Stas-Otto process.
STAS-OTTO PROCESS
A. Examination of Ether Extract of Tartaric Acid Solution
Thoroughly extract the acid aqueous solution (see process of
preparation described above) two or three times with ether,
using each time about the same quantity of solvent. Employ
a separating funnel for this purpose (Fig. n). Pour the com-
bined ether extracts into a dry flask loosely stoppered. If the
solution stands for i or 2 hours at rest, a few drops of water
usually settle out. Decant the ether solution and pour through
64
DETECTION OF POISONS
a dry filter. Slowly evaporate this solution in a small glass
dish upon a water-bath previously heated slightly above 35.
Do not have gas burning during this operation! Examine the
residue as described below. An excellent method of evaporat-
ing ether consists in setting a small glass dish (8 to 10 cm. in
PlG. II. Separating Funnels and Glass Crystallizing Dishes.
diameter) upon a hot water-bath and dropping the filtered ether
extract into it as fast as the solvent evaporates. Thus a large
quantity of extract may be evaporated in a small dish. The
advantage of this method is the ease with which the residue can
be removed for the various tests. The residue is usually quite
small and it is not advisable to have it distributed over too
large a surface.
NON-VOLATILE POISONS 65
Examine the residue from the ether extract for the following
substances :
Picrotoxin Caffeine Antipyrine
Colchicin Acetanilide Salicylic Acid
Picric Acid Phenacetine Veronal
Evaporation of the ether extract, even in the absence of
members of the group, usually leaves a more or less viscous
residue, containing tartaric and lactic acids as well as fatty,
resinous and colored substances. This is especially so in
analyses of cadaveric material. Moreover ether extracts certain
metallic salts from aqueous solutions, for example, mercuric
cyanide 1 and chloride.
First, note the general appearance and taste of the residue.
Then examine it with a microscope. Very definite conclusions
as to the presence or absence of certain substances can fre-
quently be drawn. A very bitter residue should be examined
carefully for picrotoxin and colchicin. If there is a pronounced
yellow color, the examination should include picric acid also.
Veronal is colorless and has a very bitter taste. A tasteless, or
only faintly bitter, residue probably does not contain these sub-
stances and should be examined for acetanilide, antipyrine,
caffeine, phenacetine and salicylic acid.
The residue from evaporation of the ether extract may con-
tain the following substances:
Picrotoxin. Usually a thick syrup which gradually solidifies
and becomes crystalline. Tastes intensely bitter.
Colchicin. Yellowish, amorphous residue which does not
become crystalline. Tastes intensely bitter. Dissolves in
water with a yellowish color, which increases in intensity on
addition of a few drops of dilute hydrochloric acid.
1 Ether to some extent will extract mercuric cyanide from a tartaric acid solu-
tion which is not too dilute. For instance, it will remove appreciable quantities
from 100 cc. of o.i per cent, mercuric cyanide solution, but the extraction will not
be complete. The solution after five extractions will still give a distinct test for
mercury. Ether will not remove even a trace of mercuric cyanide from o.oi per
cent, solution. To test for cyanide, add ammonium sulphide solution to the
ether residue. This will precipitate mercuric sulphide and the nitrate will con-
tain ammonium sulphocyanate (see hydrocyanic acid, page 22).
5
QQ DETECTION OF POISONS
Picric Acid. Usually appears as a syrup which gradually
solidifies and becomes crystalline. Tastes very bitter. Resi-
due intensely yellow, giving yellow aqueous solutions not in-
tensified by hydrochloric or sulphuric acid.
Acetanilide. Leaflets or flattened needles. Has a faint,
burning taste but is not bitter.
Phenacetine. Inodorous and tasteless leaflets and small
needles.
Antipyrine. Residue a syrup which is rarely crystalline.
Tastes mildly bitter. Very easily soluble in water.
Caffeine. Residue composed of shining needles frequently
in radiating clusters. Tastes mildly bitter.
Salicylic Acid. Crystallizes frequently in long needles.
Tastes harsh and at the same time sweet and acid.
Veronal. Crystalline needles having an agreeable, bitter
taste.
PICROTOXIN
Picrotoxin, CsoI^Ois, the poisonous principle of Cocculus indicus, the fruit of
Menispermum Cocculus, crystallizes from hot water in long colorless needles
melting at 190-200. It dissolves with difficulty in cold water but more readily in
hot water or ethyl alcohol. It is slightly soluble in ether but freely soluble in
chloroform, amyl alcohol and glacial acetic acid. Its alcoholic solution is neu-
tral and laevo-rotatory. Picrotoxin has a very bitter taste. It is not as readily
soluble in acids as in pure water, but is soluble in caustic alkalies and aqueous
ammonia, forming unstable, salt-like compounds which do not crystallize. Pi-
crotoxin behaves toward strong bases as if it were a weak acid. Heated to
boiling with twenty times its volume of benzene, it is decomposed into picro-
toxinin and picrotin. The former passes into solution but picrotin is almost
completely insoluble:
Cl 6 Hl8p7
Picrotoxin Picrotoxinin Picrotin
Chloroform brings about this cleavage even more easily. On the other hand,
if picrotoxinin and picrotin in molecular proportions are dissolved in hot water,
picrotoxin crystallizes out as the solution cools. Treated with bromine direct or
dissolved in water or ether, picrotoxin is first split into picrotoxinin and picrotin.
The former is immediately converted into monobromo-picrotoxinin, Ci 6 Hi B BrO 6 ,
but picrotin remains almost unchanged. Monobromo-picrotoxinin is soluble
with difficulty in water but is reduced by zinc dust and acetic acid to picrotoxinin.
Picrotin is almost non-toxic, whereas picrotoxinin has a very poisonous action.
Picrotoxin is a powerful convulsive poison, standing in its action between cicu-
toxin and strychnine.
NON-VOLATILE POISONS 67
R. Meyer and P. Bruger 1 regard picrotoxin as a complex of the two compounds,
picrotin and picrotoxinin, crystallizing together in definite but not molecular
proportion, and not as a molecularly constituted chemical compound.
Detection of Picrotoxin
1. Fehling's Test. Dissolve picrotoxin in a small test-tube,
using 10-20 drops of very dilute sodium hydroxide solution.
Add a few drops of Fehling's solution 2 and warm but do not
shake. A red or yellowish red precipitate forms and settles to
the bottom. If the ether residue, not too little of which should
be taken, fails to give a clear solution in very dilute sodium
hydroxide solution, filter through moistened paper and examine
the filtrate with Fehling's solution.
2. Anunoniacal Silver Test. Warm picrotoxin with aqueous
silver nitrate solution containing a slight excess of ammonium
hydroxide solution. The reducing action' of picrotoxin will
produce a black precipitate of metallic silver, or a dark brown
color when only traces are present.
3. Oxidation Test. Picrotoxin, treated with a little con-
centrated sulphuric acid in a porcelain dish, first becomes
orange-red and then dissolves when stirred forming a reddish
yellow solution. A drop of potassium dichromate solution will
produce a red-brown color around the margin of the drop. If
the two liquids are thoroughly mixed, there is an immediate
dirty brown color which passes into green on long 'standing.
A green color alone is without significance, since many organic substances
capable of reducing chromic acid to chromic oxide produce the same result.
4. H. Melzer's Test. 3 Put some picrotoxin upon a watch-
glass and add i or 2 drops of a mixture of benzaldehyde and
absolute ethyl alcohol. Careful addition of a drop of concen-
trated sulphuric acid will produce a distinct red color. If the
watch-glass is tilted, red streaks will run from the substance
through the liquid.
Use a freshly prepared, 20 per cent, solution of benzaldehyde in absolute ethyl
alcohol. Benzaldehyde alone gives a yellowish brown color with concentrated
1 Berichte der Deutschen chemischen Gesellschaft 31, 2958 (1898;.
2 Fehling's solution heated by itself should not give a precipitate of cuprous
oxide.
3 Zeitschrift fur analytische Chemie 37, 351 and 747 (1898).
68 DETECTION OF POISONS
sulphuric acid. Ethyl alcohol is added as a diluent to diminish this color as
much as possible. Under these conditions the solution has a light yellow color,
and the dark red tint caused by picrotoxin is very clearly denned. This red
color is unstable and, beginning at the margin, gradually fades into a pale pink
or violet. H. Kreis 1 has found that cholesterine and phytosterine 2 give similar
colors with Melzer's reagent.
5. Langley's Test. Mix picrotoxin with about 3 times the
quantity of potassium nitrate, and moisten the mixture with the
smallest possible quantity of concentrated sulphuric acid.
Then add strong sodium hydroxide solution in excess and an
intense red color will appear.
Detection of Picrotoxin in Beer
First, neutralize the beer with magnesium oxide. Then evaporate 500 cc. or
more to a syrup upon the water-bath. Digest this residue with 4 or 5 times its
volume of ethyl alcohol and evaporate the alcoholic extract. Dissolve the residue
in hot water and filter the solution through a moistened paper. Acidify the fil-
trate with dilute sulphuric acid and extract repeatedly with ether, or better with
chloroform. Evaporate these extracts and test the residue for picrotoxin.
Should the residue from the ether or chloroform be too impure, dissolve it again
in hot water, filter, evaporate and extract with ether or chloroform. To purify
picrotoxin further, precipitate colored substances from its aqueous solution with
lead acetate, filter and remove lead from the filtrate by hydrogen sulphide. The
filtrate from lead sulphide upon evaporation, or extraction with ether or chloro-
form, will give nearly pure picrotoxin. The very bitter taste of picrotoxin as
well as its strong tendency to crystallize are additional characteristics of this
substance.
COLCHICIN
Colchicin, C22H 2 5NOe, an alkaloid occurring in all parts of the meadow saffron,
Colchicum autumnale, is a yellowish, amorphous powder which is poisonous and
very bitter to the taste. It is freely soluble in water, ethyl alcohol, and chloro-
form, less so in ether and benzene, and almost insoluble in petroleum ether.
Solutions of colchicin have a more or less yellowish color which becomes more
pronounced upon addition of acids or alkalies. These solutions have very faint
basic properties. Consequently ether or chloroform, but not benzene nor petro-
leum ether, will extract colchicin from an acid, aqueous solution. Upon
evaporation of the solvent, colchicin will appear as a yellowish, sticky residue
resembling a resin or varnish. Heated with water containing sulphuric acid,
colchicin splits into colchicein and methyl alcohol. Boiling the alkaloid 1.5-2
hours with 60 parts of i per cent, hydrochloric acid will produce the same result:
C22H25N0 8 + H 2 = C 21 H 23 N06 + CH 3 .OH
Oolchicm Colchicein Methyl Alcohol
1 Chemiker-Zeitung 33, 21 (1899).
2 A substance very similar to cholesterine, and named paracholesterine or
phytosterine, is found in the seeds of certain plants. (Perkin and Kipping,
Organic Chemistry, page 608.)
NON-VOLATILE POISONS 69
On the other hand, colchicin is formed when colchicein is heated to 100 with
sodium methylate (CH 3 .ONa) and methyl iodide (CHa.I). Since colchicein on
treatment with hydriodic acid yields three molecules of methyl iodide, colchicein
as well as colchicin contains three methoxyl groups. Heated with strong hydro-
chloric acid, colchicein loses acetic acid and passes into trimethyl-colchicinic
acid. Consequently colchicein and colchicin contain an acetyl group ( CHs. CO ) .
The formula of colchicin, that is to say, of methyl-colchicein, may be written as
follows:
CH 3 0\ /NH.CO.CHs
CHsO^C 15 H 9 <
CH 3 O/ X CO.OCH 3
Detection of Colchicin
Aqueous colchicin solutions, especially in presence of dilute
mineral acids, have a yellow color. Unless the ether residue has
this characteristic, colchicin is absent.
1. Tannic Acid Test. This reagent will precipitate colchicin
from aqueous solution, if not too dilute, as white flocks. This
test, however, is not characteristic of colchicin.
2. Nitric Acid Test. Nitric acid (Sp. gr. 1.4 = 66 per cent.)
dissolves colchicin with a dirty violet color which soon changes,
when stirred, to brownish red and finally to yellow. Addition
of dilute sodium or potassium hydroxide solution, until the
reaction is alkaline, produces an orange-yellow or orange-red
color.
3. Sulphuric Acid Test. Concentrated sulphuric acid dis-
solves colchicin with an intense yellow color. A drop of nitric
acid added to such a solution produces a green, blue, violet and
finally a pale yellow tone. Excess of potassium hydroxide solu-
tion will now bring out an orange-red color. Erdmann's reagent
(see page 3 20) dissolves colchicin with a blue to violet color.
4. Hydrochloric Acid Test. Concentrated hydrochloric acid
dissolves colchicin with an intense yellow color. Add two drops
of ferric chloride solution and heat the mixture 2-3 minutes in a
test-tube. The color deepens and the solution on cooling,
especially if diluted with the same volume of water, becomes
green or olive-green. Finally shake the solution with a few
drops of chloroform. This solvent becomes yellowish brown,
or garnet-red, and the aqueous solution retains its green color.
Zeisel's reaction.
70 DETECTION OF POISONS
Purification of the Residue Containing Colchicin
To isolate as pure colchicin as possible from the yellow
residue, extract with warm water. Filter the solution and,
when cold, extract it first with petroleum ether. This will
remove fatty, resinous and colored impurities but not colchicin.
Then extract with chloroform. Or precipitate colchicin from
aqueous solution, which must not be too dilute, with tannic
acid. Collect this precipitate upon a filter and wash with cold
water. Mix the moist precipitate with freshly precipitated,
washed lead hydroxide. Dry the mixture, grind to a powder
and extract with chloroform. Evaporation of the solvent will
leave nearly pure colchicin.
PICRIC ACID
Picric acid, or 2,4,6-trinitrophenol, crystallizes from water in light yellow
leaflets and from ether in lemon-yellow, rhombic prisms. It melts at 122.5.
Though soluble in cold water with difficulty, picric acid dissolves freely in hot
water, as well as in ethyl alcohol, ether and benzene. Aqueous solutions have an
acid reaction, a very bitter taste and dye animal fibers fast
yellow. Material containing picric acid has a yellow or
C yellowish green color.
Physiological Action and Elimination. Picric acid is
OaN Y |j- NO2 q u it e an active poison. Taken internally it produces a
HC CH striking yellow pigmentation first of the conjunctiva and
then of the entire skin, usually designated as "picric acid
C icterus." Picric acid and its salts like most nitro-com-
J,~ pounds decompose the red blood-corpuscles forming met-
haemoglobin. Consequently it is a blood-poison. At the
same time it irritates the central nervous system and causes convulsions.
Finally it exercises its power of precipitating proteins in acid solution. This is
especially noticeable in those organs of the body, for example, the stomach and
QTT kidneys, which, owing to necrotic tissue changes, have an
iacid or only a faintly alkaline reaction. The organism re-
duces picric to picramic acid which does not so readily
o T_^\, ^TTT P re cipitate protein. By thus changing picric acid the
2 organism rids itself of the poison. In picric acid poisoning
HC CH tne urine has a marked red color owing to formation of
picramic acid. Some picric acid passes into the urine
unchanged. Elimination is slow. In one case (see R.
NO 2 Kobert, "Intoxikationen"), after administration of a single
Picramic acid dose of i gram of picric acid, its presence in the urine could
be recognized for 6 days. The urine was ruby-red, clear, acid and free from
albumin and bile-constituents. Picric acid was also easily detected in the feces.
NON-VOLATILE POISONS 71
Detection of Picric Acid
Material containing picric acid has a more or less yellow or
yellowish green color. Aqueous, alcoholic and ethereal solu-
tions show the same color. Finely divided animal material
should be extracted several hours under a return-condenser with
ethyl alcohol containing hydrochloric acid to decompose com-
pounds of picric acid with albumins and thus bring the acid into
solution. Filter and evaporate such an alcoholic extract upon
the water-bath. Treat the residue, which is yellow, yellowish
green, or frequently yellowish red or reddish brown, with
warm water and filter the extract. The filtrate itself may
be tested directly for picric acid, or it may first be extracted
as usual with considerable ether. The following tests may
then be applied to the residue left on evaporating the ether
extract :
i. Isopurpuric Acid Test. Gently heat (50-60) an aqueous
solution of picric acid with a few drops of saturated, aqueous
potassium cyanide solution (i : 2). The solution will become
red owing to formation of potassium isopurpurate. One milli-
gram of picric acid, dissolved in 5 cc. of water, will give a distinct
test.
Isopurpuric acid does not exist in the free state but is present in this test as
the potassium salt. Nietzki and Petri 1 regard isopurpuric acid (CgHaOjNs)
as a dicyano-picramic acid = 5-oxy-6-amino-2,4-dinitro-isophthalic nitrile;
whereas Borsche 2 considers it a dicyano-dinitro-oxy-/3-phenyl hydroxylamine:
OH OH
I II
c c
s\ /\
O 2 N C C NH 2 10 2 N C C NH.OH
I II II
NC C C CN NC C C CNj
N0 2 N0 2
Xietzki-Petri Borsche
1 Berichte der Deutschen chemischen Gesellschaft 33, 1788 (1900).
2 Ibid., 33, 2718 and 2995 (1900).
72 DETECTION OF POISONS
2. Picramic Acid Test. (a) Heat picric acid solution with
a few drops of sodium hydroxide solution and glucose. Picra-
mic acid, formed by reduction of picric acid, colors the solu-
tion deep red. Avoid excess of sodium hydroxide solution,
otherwise there will be a red color due solely to the action of the
alkali upon glucose.
(|8) The test may also be made by warming picric acid
solution with a few drops of sodium hydroxide and ammonium
sulphide solutions. This will reduce picric acid and produce a
red color.
In both reactions (a and 0) picric acid is reduced to picramic
acid, 2-amino-4,6-dinitro-phenol :
OH OH
c c
s\ /\
O 2 N C 6 2 C NO 2 2 N C 6 2 C NH 2
| || +6H= | || + 2 H 2 0.
HC CH HC CH
\4/ . \4/
C C
NO 2 NO 2
Picric acid Picramic acid
The presence of fat and other impurities materially influence
this test.
3. Dyeing Test. Dissolve the substance containing picric
acid in hot water and put white threads of wool, silk and cotton
in the solution. In a few hours (12 to 24) remove the threads
and thoroughly rinse in pure water. If picric acid is present,
the wool and silk will be dyed yellow but not the cotton. In
other words, picric acid is not fast upon vegetable fibers like
cotton. Picric acid, diluted i : 100,000, will still produce a
yellow color upon wool.
4. Ammoniacal Copper Test. Add a few drops of ammonia-
cal copper sulphate solution (copper sulphate solution and an
excess of ammonia) to an aqueous picric acid solution. A yel-
lowish green precipitate, consisting of hexagonal needles with a
polarizing action upon light, will appear. Picric acid, diluted
i : 80,000, will give this test.
NON-VOLATILE POISONS 73
ACETANILIDE
Acetanilide crystallizes in colorless and inodorous, shin-
ing leaflets. It has a faint, burning taste; melts at 113 to
114; is soluble'in 230 parts of cold water, in about 22 parts
r of boiling water and in 3.5 parts of ethyl alcohol; and is
I I j freely soluble in ether and still more so in chloroform. All
HC CH acetanilide solutions are neutral. Heated to boiling with
\/ potassium hydroxide solution (I) and also with fuming
hydrochloric acid (II), acetanilide is decomposed into its
constituents:
I. CeHs.NH.CO.CHs + KOH = C 6 H 5 .NH 2 + CH 3 .CO.OK.
II. C 6 H 6 .NH.CO.CH 3 + HC1 + H 2 O = C 6 H 5 .NH 2 .HC1 + CH 3 .COOH.
Physiological Action. Being an aniline derivative, acetanilide has the poison-
ous properties of that amine though in less degree. R. Robert (" Intoxikatio-
nen") refers to several instances of acetanilide poisoning which did not terminate
fatally. In one case a student took a teaspoonful of the drug. There was
stupor, uneasiness, marked cyanosis and lowering of the pulse. A purgative
and restorative (stimulant) were used but there was considerable exhaustion for
several days. The picture was nearly the same in the case of a man who took
2 grams of antifebrine daily for 2 days in succession.
Preparation. Boil aniline and glacial acetic acid together for several hours
under a return-condenser:
C 6 H 6 -NH 2 + CHs-COOH = C 6 H 5 -NH-CO-CH 3 + H 2 O.
Detection of Acetanilide
Ether or chloroform will extract acetanilide completely from
an acid aqueous solution.
1. Indophenol Test. Boil acetanilide with about 4 cc. of
fuming hydrochloric acid and evaporate to a few drops (about
10). Cool and add 4 cc. of saturated, aqueous carbolic acid
solution. A few drops of calcium hypochlorite solution will
produce a violet-red color. In time the color will become deeper ,
especially if the mixture is shaken. Then carefully add ammon-
ium hydroxide solution as a surface-layer which will take on a
permanent indigo-blue color.
The indigo-blue color is characteristic of acetanilide only
when preceded by the red-violet color, since a mixture of aque-
ous phenol and hypochlorite solution gives a blue color with
ammonia (see carbolic acid).
Phenacetine also gives the indophenol test.
2. Phenylisocyanide Test. Boil acetanilide with 5-6 cc.
of alcoholic potassium hydroxide solution. Cool, add 2 or 3
74 DETECTION OF POISONS
drops of choloroform and again heat. The offensive odor of
phenylisocyanide will be developed.
Potassium hydroxide decomposes acetanilide into aniline
and potassium acetate (see Reaction I above). The former
with chloroform gives phenylisocyanide.
3. Calcium Hypochlorite Test. Boil acetanilide a few min-
utes with alcoholic potassium hydroxide solution as in test 2.
Dilute with water and extract aniline with ether. This sol-
vent upon evaporation will deposit aniline as an oily liquid.
Dissolve the latter in water and test with calcium hypochlorite.
Examination of Acetanilide Urine 1
Scarcely more than traces of unaltered acetanilide appear in urine even after
large doses. The most essential change occurring in the body is oxidation of the
benzene ring which produces aceto-para-aminophenol. This like most phenols
forms a conjugate sulphuric acid and appears in the urine as a salt of aceto-para-
aminophenyl sulphuric acid:
H OH O SO, OH
A A A
HC CH HC CH HO\ HC CH
+ 0=||| + >SOo = I ||
HC CH oxidation HC CH HO/ HC CH
\/ \/ conjugation \/
c c c
NH.CO.CHj NH.CO.CHs NH.CO.CH 3
Acetanilide Aceto-p-aminophenol Aceto-p-aminophenyl sulphuric acid
To some extent also, a conjugate glycuronic acid of aceto-para-aminophenol is
formed. These compounds, heated with concentrated hydrochloric acid, give
para-aminophenol which can be detected by the indophenol test previously
described.
O.S0 2 .OH OH
c c
H/\H H /\H
J- JL + 2H 2 = H 2 S0 4 + CH 3 .COOH + | ||
HC . CH HC CH
V v
? c
NH.CO.CH 3 NH 2
p-aminophenol
Such urine, boiled a few minutes with concentrated hydrochloric acid, will
usually give the indophenol test. But the test will be more certain, if para-
* To study the behavior of acetanilide in the body, take at night 0.3 gram of
s substance at a dose twice in the course of 3 hours and examine the urine
passed in the next 12 hours.
NON-VOLATILE POISONS
75
aminophenol is first isolated. Boil a larger quantity of urine (300 to 500 ccj a
few minutes with about 10 cc. of concentrated hydrochloric acid. Then add an
excess of sodium carbonate and repeatedly extract the cool urine with large
quantities of ether. Distil or evaporate the ether. Para-aminophenol usually
appears as a reddish or brownish oil. An aqueous solution of this substance will
give the indophenol test.
PHENACETINE
Phenacetine, or p-aceto-phenetidine, crystallizes in shining leaflets, Which are
without color, odor or taste, and melts at 134 to 135. Phenacetine is soluble in
about 1400 parts of cold water, 70 parts of boiling water,
i6 parts of ethyl alcohol and freely soluble in ether and
chloroform. Its solutions are neutral. Concentrated sul-
phuric acid dissolves it without color. Phenacetine is
very closely related to acetanilide but does not give the
phenylisocyanide test.
Preparation. The gradual addition of crystallized
phenol to cold dilute nitric acid (sp. gr. i.n = 17.5 per
cent.) results in the formation of a mixture of o- and
p-nitro-phenol. Since the ortho-compound is volatile with
steam, complete separation of the two products is possible by steam distilla-
tion. The residual p-nitro-phenol is converted into its sodium salt which is
heated in sealed tube with ethyl bromide and thus changed to p-nitro-phenetol.
The latter is reduced by means of nascent hydrogen from tin and hydrochloric
acid to p-amino-phenetol, or p-phenetidine, which is then boiled with glacial
acetic acid and converted into aceto-p-phenetidine, or phenacetine:
HC
Hi
CH
L
OC 2 H 6
OH JOH JSJPNa
O^a
Br:.C 2 H 6 OC 2 H 5
A A
r
1
C
HC CH HC CH
HC CH
HC CH
1 II - 1 II -
HC CH HC CH
I !l
HC CH
- 1 II
HC CH
\/ \/
C C
C
Y
__.
1
|
HO XO 2 N 2
NO 2
NiO, j
2R 4 H:
Phenol p-Nitro-
phenol
Na salt of p-
nitro-phenol
p-Nitro-
phenetol
OC 2 H 6
OC 2 H 5
i
i
/\
^\
HC CH
HC CH
1 II
HC CH
<x /
~*
1 II
HC CH
>/
C
C
i
HNiH :
NH.CO.CHs
iHOLCO.CHs
p-Phenetidine
Phenacetine
76 DETECTION OF POISONS
Detection of Phenacetine
The extraction of phenacetine by ether or chloroform from
an aqueous tartaric acid solution is complete.
1. Oxidation Test. Boil phenacetine for several minutes
with 3 cc. of concentrated hydrochloric acid. Dilute with
10 cc. of water and filter when cold. A few drops of chromic
acid solution added to the filtrate will gradually produce
a ruby-red color. Strong chlorine water may be substituted
for chromic acid.
2. Indophenol Test. Boil phenacetine i or 2 minutes with
about 2 cc. of concentrated hydrochloric acid. Dilute with
water and add a few cc. of aqueous carbolic acid solution.
Filter the solution when cold. If a few drops of freshly pre-
pared calcium hypochlorite solution are added, the filtrate will
have a fine carmine-red color. Addition of ammonium hydrox-
ide solution in excess will change this color to violet-blue.
Freshly prepared chlorine water, or 3 per cent, chromic acid
solution, may be substituted for hypochlorite solution as an
oxidizing agent.
3. Autenrieth-Hinsberg Test. 1 (a) With Dilute Nitric Acid.
Heat phenacetine to boiling with a few cc. of dilute nitric
acid (10 to 12 per cent.). It is soluble and gives an intense
yellow to orange-red color. As the solution cools, if sufficiently
concentrated, nitro-phenacetine 2 will crystallize in long, yellow
needles which melt at 103. This test is delicate, and char-
acteristic of phenacetine, especially when nitro-phenacetine can
be obtained in crystals and its melting-point determined. It
serves to distinguish phenacetine from acetanilide and anti-
pyrine, both of which give colorless solutions when warmed with
dilute nitric acid.
(6) With Concentrated Nitric Acid. A few drops of con-
centrated nitric acid poured upon phenacetine produce a yellow
1 Archiv der Pharmacie 229, 456 (1891).
1 The structural formula of mono-nitro-phenacetine is as follows:
/OC 2 H 6 i
3 \NH(C 2 H 3 0) 4
NON-VOLATILE POISONS 77
to orange-red color. Part of the phenacetine is dissolved with
the same color and heat completes the solution. Nitro-phen-
acetine crystallizes as the solution cools.
SALICYLIC ACID
COOH Salicylic acid, or ortho-oxy-benzoic acid, crystallizes in long,
| white needles soluble in about 500 parts of cold and in 15
C parts of boiling water; and freely soluble in ethyl alcohol, ether,
<f\ chloroform and carbon disulphide. It has a peculiar taste
j II which is sweetish, acidulous and rather acrid. It melts at 157.
HC CH Heated carefully, salicylic acid will sublime in fine needles
\/ without decomposition. A little of the acid may show this
behavior even upon the water-bath. If heated quickly, salicylic
acid is decomposed in part into phenol and carbon dioxide.
/COOH
C 6 H 4 < = C 6 H 5 -OH + C0 2 .
X OH
Concentrated sulphuric acid dissolves pure salicylic acid without color and
without decomposition. The lead and silver salts of this acid are soluble in water
with difficulty. Consequently lead acetate will precipitate lead salicylate,
/ /C00\
(C 6 H 4 <
\ X OH /,
Pb, from neutral solutions. This salt is white, crystalline and
soluble in hot water. It crystallizes unchanged as the hot solution cools. Silver
nitrate precipitates white silver salicylate.
R. Schmitt's Method of Preparation. Dry sodium phenolate is kept cool and
saturated in an autoclave under pressure with carbon dioxide (a). The sodium
phenyl-carbonate undergoes molecular rearrangement, when heated at 120-130,
and becomes isomeric sodium salicylate (/3):
/0 ONa /0 Na Molecukr /OH (i)
(a) C +| = C = O (/3) rearrangement C 6 H 4
\) C 6 H 5 ^OCsHs gives: \COONa (2)
Sodium phenyl- Sodium salicyl-
carbonate ate
Detection of Salicylic Acid
i. Ferric Chloride Test. Addition of ferric chloride solution
to a solution of salicylic acid or salicylates produces a blue-
violet color. If the solution is very dilute, the color is more of a
red-violet. Hydrochloric acid changes the violet color to
yellow. An excess of the 'reagent affects the delicacy of the test.
This test fails in presence of mineral acids, caustic alkalies and alkaline
carbonates.
78 DETECTION OF POISONS
2. Millon's Test. If an aqueous salicylic acid solution is
warmed with Millon's reagent, a deep red color will appear.
3. Bromine Water Test. This reagent in excess produces a
yellowish white, crystalline precipitate even with very dilute
salicylic acid solutions. The compound thus formed is tri-
bromo-phenyl hypobromite (see page 28).
OBr
C
/OH Brcf\Br
C 6 H 4 + 4Br 2 = CO 2 + 4HBr + | ||
\COOH yCH
Br
4. Melting-Point Test. If the quantity of salicylic acid is
not too small, dissolve the ether residue in very little hot water,
shake the hot solution with a little animal charcoal and filter.
Cool the nitrate, dry the crystals and determine the melting-
point (157).
Separation of Salicylic Acid from Simple Phenols
When phenols like carbolic acid or the cresols are present,
the above tests from i to 3 prove nothing as far as salicylic
acid is concerned. If these compounds are present, add suffi-
cient sodium carbonate solution to render the ether residue
alkaline and extract the solution with ether. This solvent
will take up the phenols and salicylic acid will remain in the
water as the sodium salt. Withdraw the aqueous solution
from the separating funnel, acidify with dilute hydrochloric
or sulphuric acid and extract salicylic acid with ether.
Directions are given elsewhere (see page 250) for the detection
of salicylic acid in beer, milk, urine, fruit juices, meat and meat
preparations, as well as in maltol.
Quantitative Estimation of Salicylic Acid as Tribromo-phenyl Hypo-
bromite
Place the aqueous solution of salicylic acid in a glass-stop-
pered flask, add an excess of saturated bromine water and shake.
The acid is completely precipitated as tribromo-phenyl hypo-
NON-VOLATILE POISONS 79
bromite. At the end the solution should be reddish brown.
It should stand 12-24 hours, and be shaken frequently. Collect
the precipitate of tribromo-phenyl hypobromite in a weighed
Gooch crucible and dry to constant weight in a vacuum desic-
cator over sulphuric acid. The quantity of salicylic acid may
be calculated from the weight of precipitate as follows:
CeHaB^OrCrHeOs = Wt. of precipitate : x
409.86 138.05 obtained
Detection of Salicylic Acid in Urine 1
Salicylic acid forms a conjugate with glycocoll, supplied by the organism,
and is changed in the human body, in part at least, into salicyluric acid,
/OH (i)
C 6 H 4 <
X CO.NH.CH 2 .COOH (2)
This is eliminated in urine with unaltered salicylic acid. Such urine gives a
violet color with ferric chloride solution. Both salicylic and salicyluric acids
give this test. To decompose salicyluric acid into its constituents, heat the acid
half an hour with fuming hydrochloric acid under a return-condenser.
To isolate unchanged salicylic acid, acidify 500 to 1000 cc. of urine with hydro-
chloric acid and repeatedly extract with ether. Remove the ether from the
aqueous solution in a separating funnel and shake vigorously with excess of
sodium carbonate solution. Salicylic acid passes into the aqueous solution.
Withdraw the aqueous solution, which is alkaline, acidify with dilute hydro-
chloric acid and extract with ether which upon evaporation usually deposits
the acid in a crystalline condition. Purify the residue by recrystallization from
water, using animal charcoal to remove color. Salicylic acid is rapidly taken up
by all mucous surfaces and quickly absorbed. Elimination by way of the urine
usually begins within the first half hour and is complete in 3 days.
VERONAL
Veronal, or barbital (name adopted by the Federal Trade Commission for the
substance formerly sold under the protected name "veronal"), is C-diethyl-
barbituric acid, C-diethyl-malonyl-urea, CsHuOsNj. It crystallizes from hot
water in large, colorless, spear-shaped crystals melting at 191 (corrected) and
C 2 H S \ /CO NH\ is soluble in 146-147 parts of water at 20 and in
/C<^ /CO 15 parts at 100. Veronal is also freely soluble in
CzHs' ^CO NH/ not et h y i a l C ohol and in acetone. It dissolves with dif-
ficulty in cold ether. An aqueous veronal solution has a bitter taste and shows
a very faint acid reaction with sensitive blue litmus paper. Veronal readily dis-
1 To study the behavior of salicylic acid in this connection, take i to 1.5 grams
of sodium salicylate at night in the course of several hours and examine, as de-
scribed, the urine passed in the next 12 hours.
gO DETECTION OF POISONS
solves in caustic alkalies, ammonia and in calcium or barium hydroxide solu-
tion. From such solutions, provided they are not too dilute, acids reprecipitate
veronal in a crystalline condition. Of the veronal salts the sodium salt, C 8 HnO 5 -
N 2 Na, crystallizes best. It may be prepared by dissolving veronal in the
calculated quantity of caustic soda solution free from carbonate, and then evapo-
rating this solution with exclusion of carbon dioxide, or adding ethyl alcohol
until turbidity appears. In both cases the sodium salt of veronal separates as
splendid shining crystals.
Preparation by E. Fischer and A. Dilthey 1
(a) From diethyl-ethylmalonate by condensation with urea in presence of
sodium ethylate:
NajOCjH.
C 2 H 8 CO lOCg'HJNiH C 2 H 6 CO N Na
\C<^ ' 5 CO = / c <( / co + 3C 2 H 6 .OH.
C 2 H 6 CO ipCaHsHjNH C 2 H 6 CO NH
Diethyl-ethymaionate Urea Na salt of veronal
Dissolve metallic sodium (32 parts) in absolute ethyl alcohol (600 parts) and
when cold add diethyl-ethylmalonate (100 parts). Dissolve in this mixture with
heat finely powdered urea (40 parts). Heat 4-5 hours in an autoclave at 105-
108. The sodium salt of veronal is precipitated even from the hot solution as a
colorless, crystalline mass. Cool, filter with suction and wash with ethyl alcohol.
Dissolve the crystals in water and acidify with concentrated hydrochloric
acid. Veronal thus precipitated is pure when recrystallized from water.
(b) From diethyl-malonyl chloride by condensation with urea:
c 2 H 6 co |cFHJNH c 2 H 5 co NH
^>C<^ yCO = 2HC1 + / C <T / CO
C 2 H 6 CO jcFJiJNH C 2 H 6 CO NH
Heat diethyl-malonyl chloride (3 parts) on the water-bath for 20 hours with
finely powdered, dry urea (2 parts). Considerable hydrochloric acid is given
off toward the end and a solid mass finally remains which yields pure veronal
upon crystallization from hot water. The yield is 70 per cent, of the theoretical
amount.
Physiological Action. Veronal does not cause decomposition of the blood and
in the usual medicinal doses (0.5-1 gram) does not appear to act strongly upon
the heart. Cumulative action has been noted only in rare instances. In large
doses, however, veronal may cause serious intoxication with fatal termination.
Death resulted in the case of a man in Holzminden who had taken 10 grams of
veronal by accident. There are also on record two other suicidal cases, one from
ii grams and the other from 15 grams of this hypnotic. There was loss of con-
sciousness and contraction of the pupils in the second case. Atropine caused
dilatation of the pupils but otherwise was of no avail. Death ensued in 20 hours.
1 Annalen der Chemie und Pharmazie, 335, 334 (1904).
NON-VOLATILE POISONS 81
Detection of Veronal
In examining cadaveric material (liver, spleen and kidneys)
from a man, who had taken veronal, thinking it was a remedy
for tape- worm (kamala), G. and H. Frerichs 1 isolated small
quantities of this drug. Following the Stas-Otto process, they
extracted the aqueous tartaric acid solution with ether and evap-
orated the ether extract. They recrystallized the residue from
a Httle hot water, using animal charcoal to remove color, and
identified the crystals obtained as veronal by the following tests :
1. The aqueous solution of the crystals had a faintly acid
reaction.
2. The crystals were soluble in sodium hydroxide solution
and ammonia and were reprecipitated when the solutions were
acidified with dilute hydrochloric acid.
3. The melting-point of the crystals was 187-188. On
mixing the substance with pure veronal, they obtained the
same melting-point. 2
4. Presence of nitrogen was shown by fusing the crystals in a
dry test-tube with metallic sodium, cooling the melt, dissolving
in water and testing for sodium cyanide by the Prussian blue
reaction (see page 2 2) .
5. The crystals were heated in a dry test-tube and sublimed.
They were then compared with ciystals known to be pure
veronal and found identical.
Detection of Veronal in Urine
E. Fischer and J. v. Mering, 3 and also B. Molle and H. Kleist, 4
have found that most of the veronal leaves the human body
unchanged and is present in the urine to the extent of 70-90
per cent. Consequently in veronal poisoning the urine should
be examined first. Concentrate a considerable quantity on
1 Archiv der Pharmazie 244, 86-90 (1906).
* A mixture of two organic substances, having the same melting-point but not
being identical, will show a melting-point lower than that of either substance
taken by itself. But obviously a mixture of identical substances will show no
depression of melting-point whatever.
3 Die Therapie der Gegenwart 45, 1904.
4 Archiv der Pharmazie 242, 401 (1904).
82 DETECTION OF POISONS
the water-bath 1 to one-fifth its volume and extract several
times with ether, using a large volume at each extraction be-
cause veronal is not very soluble in this solvent. The residue
left after distilling the ether is usually quite dark in color.
Dissolve in as little hot water as possible, boil the solution 15
minutes with animal charcoal and filter. Cool the nearly
colorless filtrate with ice and veronal will crystallize in colorless
needles melting at 191 (corrected).
E. Fischer and v. Mering recovered from 5 days urine, after administration
of 4 grams of veronal during 2 days, 2.49 grams = 62 per cent, of the quantity
used. The method therefore is not absolutely quantitative, nor is the elimination
of veronal complete after 5 days. The crystals obtained should be proved
conclusively to be veronal by the tests described.
Molle and Kleist first add lead acetate solution to the urine as long as it causes
a precipitate, filter, remove lead from the nitrate by hydrogen sulphide and filter
from lead sulphide. Hydrogen sulphide is expelled with heat and the urine,
after dilution with twice its volume of water, is boiled with animal charcoal.
The filtrate, after concentration on the water-bath to a small volume, is cooled,
saturated with sodium chloride and extracted 3 times with ether. The filtered
ether solution on distillation leaves nearly pure veronal.
ANTIPYRINE
Antipyrine, or i-phenyl-2, j-dimethyl-isopyrazolone, C u Hi 2 ON2, forms
monoclinic, tabular crystals having a faintly bitter taste and melting at 113.
/ \ jj p jj pjT / \ One part of antipyrine is soluble in less than i
1 1 | part of cold water, in about i part of ethyl alcohol,
HC N C 6 H 5 (i) i part of chloroform and in about 50 parts of
\f ether. An aqueous antipyrine solution has a
Q neutral reaction, although this compound is a
base and forms crystallizable salts with acids.
Preparation. Antipyrine is formed directly by heating /3-phenyl-methyl-
hydrazine and aceto-acetic ester:
CH,--C|0 HJ N-CH 3 (3) CH,-C-N-CH 3 (2)
HCJHi iHiN-C 6 H s = HC N-C 6 H 5 (i) + H 2 O + C 2 H 5 .OH.
0b C s H 5 i C
o o
Detection of Antipyrine
Ether extracts only small quantities of antipyrine from a
solution containing much tartaric acid. Ether, or better
chloroform, extracts by far the greater part of the antipyrine
1 Fischer and v. Mering evaporated the urine under diminished pressure.
NON-VOLATILE POISONS 83
when the solution has been made alkaline. Antipyrine differs
from most alkaloids in being more soluble in water. To detect
antipyrine, dissolve in a little water the residue left on evapo-
rating, the ether solution, filter and apply the following tests:
1. Ferric Chloride Test. Add i or 2 drops of ferric chloride
solution to an aqueous antipyrine solution. It will produce a
deep red color which can be seen even in a dilution of i : 100,000.
2. Tannic Acid Test. Tannic acid solution produces an
abundant, white precipitate, when added to an aqueous anti-
pyrine solution.
3. Finning Nitric Acid Test. Dissolve antipyrine in a few
drops of water and add i or 2 drops of fuming nitric acid. The
solution will be green. If this solution is heated to boiling,
another drop of nitric acid will produce a red color. Two cc.
of antipyrine solution (i : 200) will give this test distinctly.
4. Nitroso-antipyrine Test. Add a few drops of potassium
or sodium nitrite solution to an aqueous antipyrine solution
and then dilute sulphuric acid. A green or blue color will
appear. A few drops of acetic acid may be substituted for
sulphuric acid but the solution must be heated. If the anti-
pyrine solution has been concentrated, green crystals of nitroso-
antipyrine (CnHu(NO)ON 2 ) will separate after some time.
Detection of Antipyrine in Urine
Part of the antipyrine passes unchanged into the urine but some is also present
as oxy-antipyrine-glycuronic acid, a direct test for which may be made by means
of ferric chloride solution. When the urine is highly colored, render a consider-
able quantity alkaline with sodium hydroxide solution or ammonia and extract
with chloroform. Dissolve the residue left by chloroform in a little water and
test for antipyrine with ferric chloride solution and with fuming nitric acid.
CAFFEINE
Caffeine (theine) or i,3,7-trimethyl-2,6-dioxy-purine
crystallizes in white, shining needles. It is soluble in 80 parts of water, giving
/ \ CHs _ N _ CO(6) a colorless solution with a neutral reaction and
/CH 3 (7) a faint, bitter taste. Caffeine is quite easily
(2) OC C N<(^ soluble in hot water (i : 2). It requires for
I II ^^^ solution nearly 50 parts of ethyl alcohol, only 9
parts of chloroform and is only slightly soluble
in ether. In crystallizing from hot water caffeine combines with i molecule of
water, a part of which it loses upon exposure to air and all when dried at 100.
84 DETECTION OF POISONS
Caffeine is only very slightly soluble in absolute ethyl alcohol, benzene and
petroleum ether. It melts at 230, but somewhat above 100 begins to volatilize
in small quantity and at 180 to sublime without leaving a residue. Concen-
trated sulphuric and nitric acids dissolve it without color. Caffeine is a very
weak base and its salts are decomposed by water. Therefore, caffeine can be ex-
tracted at least partially by ether, or better by chloroform, from an aqueous tar-
taric acid solution. The relation existing between caffeine and uric acid is quite
apparent when the products, formed by oxidizing these two substances with
potassium chlorate and hydrochloric acid, are compared. Oxidation of uric
acid yields alloxan and urea; caffeine gives dimethyl-alloxan and monomethyl-
urea.
C H 3 N CO; CH 3 N CO NH CH 3
OC C ! N^-CH 3 + (H,0) + 2 = OC CO + CO
I II i >CH I I I
CH 3 N C ; N^ CH 3 N CO NH 2
Caffeine Dimethyl- Monomethyl-
alloxan urea
Fate of Caffeine in Human Metabolism. Only a very small part of the caffeine
taken into the body passes through unchanged and appears in the urine. About
10 per cent, appears in the urine as decomposition products. The remainder
may be changed into normal end-products of human metabolism. Most of the
nitrogen of caffeine is eliminated as urea. A very important fact is the cleavage
of methyl groups with formation of the first decomposition products of caffeine,
namely, dimethyl- and monomethyl-xanthines. Of the mono methyl xanthines,
7-monomethyl-xanthine is formed especially. Of the dimethyl-xanthines,
paraxanthine = i,7-dimethyl-xan thine is found. Both of these compounds
appear in urine after administration of caffeine. Paraxanthine is isomeric
with theophylline, or i,3-dimethyl-xanthine, and with theobromine, or
3 , 7-dimethyl-xanthine.
The structural formulae of these cleavage-products of caffeine in animal
metabolism are as follows:
HN CO (i) CH 3 .N CO
OC C N/ OC C NH
| II \r>
CH
(3 ) CH..N-C-N*
7-Methyl-xanthine Theophylline
(i) CH 3 .N CO HN CO
II II
OC C-N.CH 3 ( 7 ) OC C N.CH 3 ( 7 )
>CH \CH
HN C N^ ( 3 ) CH 3 .N C N^
Paraxanthine Theobromine
Detection of Caffeine
Ether will extract more caffeine from an aqueous alkaline
solution than from an aqueous tartaric acid solution. Since
NON-VOLATILE POISONS 85
caffeine dissolves with some difficulty in ether, but more easily
in chloroform, the latter solvent is usually employed after the
solution has been made alkaline with ammonia. After dis-
tillation of solvent, caffeine appears in concentric clusters of
long shining needles. In an analysis by the Stas-Otto method
caffeine will appear in all three extracts.
1. Oxidation Test. Pour a few cc. of saturated chlorine
water 1 over caffeine and evaporate the solution to dryness upon
the water-bath. A reddish brown residue will remain. If a
few drops of ammonium hydroxide solution are added, a fine
purple-red color will immediately appear. This test may be
made by covering the dish containing the residue with a glass
plate moistened with a drop of strong ammonia. Or two
matched watch-glasses may be used, the material containing
caffeine being evaporated to dryness with chlorine water upon
one glass which is then placed for a short time upon the other
glass containing a drop of strong ammonia.
This test, known as the murexide reaction, is also given by
xan thine, theobromine, i- and y-monomethyl-zanthine and
paraxanthine, especially if made as described by E. Fischer. 2
Heat the material to boiling in a test-tube with strong chlor-
ine water, or with hydrochloric acid and a little potassium
chlorate, evaporate the liquid to dryness in a dish and moisten
the residue with ammonia.
2. Tannic Acid Test. This reagent, added to an aqueous
caffeine solution, causes a heavy white precipitate which is
soluble in an excess of the acid. This test is not characteristic
of caffeine.
B. Examination of Ether Extract of Alkaline Solution
(Most of the alkaloids appear here)
Add enough sodium hydroxide solution to the acid solution
separated fiom ether to make it strongly alkaline. The alkali
1 A convenient method of preparing a saturated, aqueous chlorine solution is
to heat potassium chlorate with hydrochloric acid and pass the chlorine into a
small quantity of water.
2 Berichte der Deutschen chemischen Gesellschaft 30, 2236 (1897).
86 DETECTION OF POISONS
will liberate alkaloids from their salts and combine with mor-
phine and apomorphine, if present. Thoroughly extract this
alkaline solution with about the same quantity of ether. This
solvent will dissolve all alkaloids except morphine, apomorphine
and narceine. Separate the ether from the aqueous solution
and again extract with a fresh quantity of ether. In certain
cases 3 or 4 such extractions may be required. Pour the ether
extracts into a dry flask, stopper loosely and set aside for i or
2 hours. A few drops of water always settle to the bottom of
the flask. Carefully decant the ether and pour through a dry
filter. Evaporate the nitrate with gentle heat in a glass dish
(8 to 10 cm. in diameter). Let the last part of the ether solution
evaporate spontaneously. If small globules having a strong
odor appear, the residue must be examined for coniine and
nicotine. If there is no trace of these volatile alkaloids, gently
heat the residue upon the water-bath to expel water left by
evaporation of the ether. Remove the dish from the water-
bath as soon as this has been accomplished. It is not advisable
to heat the residue too long, as it tends to become viscous.
This residue, obtained by extracting the alkaline solution with
ether, may contain any alkaloid except morphine, apomorphine
and narceine. It should be examined for
Coniine Atropine Hydrastine
Nicotine Scopolamine Pilocarpine
Aniline Cocaine Quinine
Veratrine ' Physostigmine Caffeine
Strychnine Codeine Antipyrine
Brucine Narcotine Pyramidone.
First, note the general appearance of the residue and then
examine with the microscope. Taste it cautiously. Certain
alkaloids may be recognized beforehand by this test. Special
tests should then be made at once. The various alkaloids
appear in the residue as follows :
Strychnine. Fine needles having an exceedingly bitter taste.
Brucine. Usually a white, amorphous powder having a
very bitter taste.
NON-VOLATILE POISONS 87
Veratrine. Usually an amorphous powder having a sharp,
burning taste.
Atr opine and Quinine. A varnish which is resinous and
sticky. Rarely crystalline or in the form of a powder.
Codeine. A thick, viscous syrup which after a time becomes
solid, especially if stirred with a glass rod, and frequently
crystalline.
Caffeine. Long, silky needles having a faintly bitter taste.
These are frequently concentrically arranged.
Antipyrine. A syrup which gradually becomes crystalline,
especially if stirred. It has a mild, bitter taste and dissolves
very easily in water. 1
Pyramidone. Usually as fine needles which have a faintly
bitter taste. It is easily soluble in water.
Frequently ether leaves only a slight, tasteless residue. In
that case alkaloids are absent. Such residues often consist
of fat, resinous matter, or traces of nitrogenous substances
(Peptones and their cleavage-products? Creatinine?). Parts
of a cadaver, even when quite fresh, usually give small residues
at this point. Alkaloids may be absent and every step in the
process may have been performed with the greatest care. To
be quite sure that alkaloids are absent, dissolve a portion of the
residue in water containing a drop of dilute hydrochloric acid.
Filter, if necessary, and distribute this solution upon several
watch-glasses. Test with the following alkaloidal reagents:
Mercuric Chloride, Picric Acid,
lodo-Potassium Iodide, Tannic Acid,
Potassium Mercuric Iodide, Phospho-Molybdic Acid,
Potassium Bismuthous Iodide, Phospho-Tungstic Acid. .
Unless these reagents give distinct and characteristic pre-
cipitates, alkaloids are absent. It is advisable in every instance
1 Most of the alkaloids are only slightly soluble in cold water. Some cannot be
detected satisfactorily by purely chemical means. Others have no characteristic
tests. Such substances should not be selected for laboratory practice. They
may cause beginners to think that the experienced toxicologist relies upon similar
uncertain methods when he seeks to identify an alkaloid in an actual analysis.
88 DETECTION OF POISONS
to make this preliminary test for alkaloids. Only a small por-
tion of material is required and these general reagents show even
traces of alkaloids.
To exclude mistakes and oversights in toxicological analysis, dissolve the ether
residue, should it be very small, in a few cc. of very dilute hydrochloric acid
(about i per cent, of HC1). Evaporate this solution upon the water-bath and
dissolve the residue in a little water. Inject this solution from a hypodermic
syringe into the lymph-sac on the back of a small but lively frog. If the frog
shows no sign of poisoning in the course of several hours, it is quite likely that
the residue does not contain any very poisonous alkaloid.'
In making special tests for alkaloids, distribute the residue
upon several watch-glasses, using a platinum or nickel spatula
or a small penknife. Or dissolve the residue in a little hot ethyl
alcohol, filter the solution, distribute upon watch-glasses and
evaporate at a gentle heat. R. Mauch 1 dissolves the residue
in 75 per cent, aqueous chloral hydrate solution and uses this
solution in testing for alkaloids. (The details of this method
will be found on page 251.) .
Purification of the Alkaloidal Residue
If alkaloids are contaminated with greasy, resinous or fatty
substances, many of the tests will either fail entirely or give
uncertain results. In this case the residue must be purified
in one of two ways.
1. Thoroughly mix the residue with cold water containing
hydrochloric acid. Filter to remove insoluble matter (fatty
or resinous substances), add sodium hydroxide solution to the
filtrate until alkaline and extract with ether. The alkaloids
obtained by evaporating the solvent are usually quite pure.
2. Or dissolve the residue in hot amyl alcohol, extract this
solution with a few cc. of very dilute sulphuric acid and with-
draw the acid solution from the separating funnel. Amyl
alcohol will retain greasy and colored impurities, and the alka-
loids will be in the aqueous solution as sulphates. Add sodium
'Richard Mauch (Mittheilungen aus dem Institut des Herrn Prof. Dr. E.
Schaer i i Strassburg), "Festgabe des Deutschen Apotheker-Vereins," Strassburg,
NON-VOLATILE POISONS 89
hydroxide solution in excess and extract with ether. This
method of purifying the alkaloidal residue is especially recom-
mended, when there is considerable coloring matter.
W. H. Warren and R. S. Weiss 1 have suggested picrolonic acid 2 as a means of
purifying alkaloids. An alkaloid like strychnine, whose picrolonate is very in-
soluble, may be precipitated from aqueous solution and thus separated from other
substances which prevent purification. The precipitated picrolonate may be
collected on a filter, washed with water and then warmed with dilute sulphuric
acid which discharges the bright yellow color of the picrolonate causing the alka-
loid to pass into solution and precipitating pale yellow picrolonic acid. By
extracting with acetic ether, in which picrolonic acid is especially soluble, the
aqueous solution of the alkaloid is left colorless. Neutralization with sodium
hydroxide solution and extraction with ether will give a very pure alkaloid.
CONIINE
Coniine, a-normal-propyl-piperidine, CgHnN, occurs in all parts of spotted
hemlock (Conium maculatum) together with n-methyl-coniine, conhydrine,
pj 2 7-coniceine and pseudo-conhydrine. It is a color-
C less, oily, very poisonous liquid which becomes
yellowish or brown in contact with air and is par-
dally resinified. It is slightly soluble in cold but
*CH CH CH CH even ^ ess soluble in hot water. Coniine is miscible
\/ with ethyl alcohol, ether, chloroform and benzene in
all proportions. The unpleasant, narcotic odor of this
alkaloid, sometimes said to resemble that of mouse
urine, is more intense than the odor of nicotine. Coniine as it occurs in nature
is dextro-rotatory, 3 ()D = +18.3, and rather a strong base. Heated with
acetic anhydride, it forms acetyl-coniine :
H C *
C 8 H 16 N.CO.CH 3 + CH,COOH;
Shaken with benzoyl chloride and sodium hydroxide solution, it forms benzoyl-
coniine:
cS*CoS + NaOH = C8H 16 N.CO.C 6 H 8 + NaCl + H 2 O;
and with nitrous acid nitroso-coniine:
CsH 16 N.NO + H 2 O.
All these reactions show that coniine is a secondary base.
1 The Journal of Biological Chemistry, 3, 330 (1907).
2 For the preparation of this reagent, see page 320.
3 The optical activity of this alkaloid is occasioned by the presence of the asym-
metric carbon atom marked with an asterisk in the structural formula.
90 DETECTION OF POISONS
Detection of Coniine
The alkaloidal reagents especially delicate with coniine are:
iodo-potassium iodide (1:8000), phospho-molybdic acid
(1:5000), potassium mercuric iodide (1:8000) and potassium
bismuthous iodide (1:5000). Gold and platinum chlorides
fail to precipitate coniine when the concentration is less than
i : 100; whereas they will precipitate nicotine when the concen-
tration of the solution is as low as i: 10,000 and 1:5000.
When coniine is present, the residue left by the ether solution
has the characteristic odor of this alkaloid. The two following
tests should then be made:
1. Solubility Test. Dissolve a drop of coniine in just enough
cold water to give a clear solution. Gently heat the solution
and it will become milky, because coniine is more easily soluble
in cold than in hot water. A coniine solution which is milky
when hot becomes clear on cooling. Aqueous coniine solutions
have an alkaline reaction. Test the solution with red litmus
paper.
2. Crystallization Test. Put a little coniine upon a watch-
glass, or glass slide, and add i or 2 drops of hydrochloric acid.
Evaporate to dryness and coniine hydrochloride (C 8 Hi 7 N.HCl)
will remain. Immediately after evaporation examine this, resi-
due with a microscope magnifying about 200 times. 1?he color-
less or faintly yellow crystals are needle-like, or columnar and
frequently grouped in star-shaped clusters. They show the
play of color characteristic of doubly refractive substances.
NICOTINE
Nicotine, CioHuNz, is a colorless hygroscopic liquid which soon turns yellow
and then brown upon exposure to air and in time becomes resinous. It is miscible
with water in all proportions (distinction from coniine)
C CH 2 CH an( * * reel y sol uble in ethyl alcohol, ether, amyl alcohol,
/\ | | benzene and petroleum ether. Ether extracts nicotine
0C CHa CH 2 from aqueous solution. It has a sharp, burning taste
TTP PTT TM an( ^ stron S odor of tobacco especially when warm.
\^ Chemically pure nicotine is said to be almost inodorous.
N CH 8 ^be so-called tobacco odor is developed after the alka-
loid has been for some time in contact with air. The
free alkaloid is strongly Isevo-rotatory, []D = -161.55, but its salts are dextro-
rotatory.
NON-VOLATILE POISONS 91
Constitution. Nicotine is a rather strong di-acid, ditertiary base and forms
well-crystallized salts with one or two equivalents of acid. Like ditertiary bases
it combines with two molecules of methyl iodide 1 forming a di-iodo-methylate,
CioHuNz.sCHsI. Oxidized with chromic acid, nitric acid or potassium per-
manganate, nicotine is converted into nicotinic acid, or /3-carboxy-pyridine.
This shows that nicotine is a pyridine derivative having a side-chain in the /3-
position with respect to the pyridine nitrogen.
H H
C CH 2 CH 2 C
' S\ I I S\
HC /3C CHa CH 2 HC C COOH
I I! \/ I II
HC CH N gives on oxidation HC CH
\/ I . \/
N CH 3 N
Nicotine Nicotinic acid
This formula for nicotine proposed by Pinner was confirmed several years later
by Ame" Pictet's synthesis of this alkaloid.
Physiological Action. Nicotine is one of the most powerful poisons and
scarcely inferior to hydrocyanic acid in toxicity and rapidity of action. It ap-
pears to be toxic to all classes of animals. It is absorbed from the tongue, the
eye and the rectum even in a few seconds and from the stomach somewhat more
slowly. Absorption of nicotine is also possible from the outer skin. Elimina-
tion takes place through the lungs and kidneys. In concentrated form nicotine
is a local irritant, though, owing to the rapidity of its toxic action, it does not
behave like a true corrosive nor does it cause inflammation of the mucous lining
of the stomach even after a lethal dose. Nicotine, after causing stimulation
for a brief period, then paralyzes the central nervous system and spinal cord,
finally affecting various organs such as the heart, eyes and intestinal tract. Its
poisonous influence probably extends to all parts of the brain, medulla oblongata
and spinal cord. Huchard states that nicotine causes a general convulsion of
the circulatory system which is apparent in chronic nicotine poisoning. In
chronic tobacco poisoning the general condition of health is disturbed and quite
frequently the eyes are affected. In acute nicotine poisoning death ensues from
paralysis of the respiratory center. An action upon the heart is also always in
evidence even in non-fatal cases.
1 In methyl iodide as well as in other alkyl haloids we have an excellent
means of recognizing the tertiary nature of a nitrogen base. Like trimethylam-
ine, tertiary cyclic amines, as pyridine and quinoline, also give similar iodo-
methylates which are ammonium iodides with quinquivalent nitrogen:
H H
CH 3 \ni rS 3 \v
CHAN + CH 3 .I = N.I;
Trimethylamine
C
HC CH
C
HC CH
1 II -
HC m CH
h CH 3 .I = | ||
HC CH
N
Y
Pyridine
H/\
92 DETECTION OF POISONS
Detection of Nicotine
Ether or low-boiling petroleum ether will extract nicotine
from an aqueous alkaline solution. Spontaneous evaporation
of the solvent leaves the alkaloid as an oily liquid having the
odor of tobacco and a strong alkaline reaction. General alka-
loidal reagents will precipitate nicotine from quite dilute solu-
tions, in which respect this alkaloid is very different from coniine.
Phospho-molybdic acid and potassium bismuthous iodide
precipitate nicotine even in a dilution of ,1:40,000; potassium
mercuric iodide in 1:15,000; gold chloride in i: 10,000; and
platinum chloride in i : 5000.
1. Crystallization Test. Evaporate nicotine on a watch-
glass with a few drops of concentrated hydrochloric acid. This
will yield a yellow, varnish-like residue which microscopic ex-
amination will show to be entirely amorphous (distinction
between nicotine and coniine). If kept for a long time in a
desiccator over sulphuric acid, it will become indistinctly
crystalline.
2. Roussin's Test. Dissolve a trace of nicotine in ether,
using a dry test-tube. Add to this solution about the same
volume of ether containing iodine. Stopper and set the test-
tube aside. The mixture will become turbid and deposit a
brownish red resin which will gradually become crystalline.
After some time, ruby-red needles with a dark blue reflex will
crystallize from, the ether. These are "Roussin's crystals."
If nicotine is old or resinous, it will not as a rule give these
crystals.
3. Melzer's Test. 1 If a drop of nicotine is heated to boiling
with 2-3 cc. of epichlorohydrin, 2 the mixture becomes dis-
tinctly red. This test applied to coniine causes no color.
1 Zeitschrift des allgemeinen Oesterreichen Apotheker-Vereins 54, 65.
CH 2 Cl - CH \
2 Epichlorohydrin, | \Q, prepared by the action of i mol. of
CH2
caustic alkali on a-dichlorohydrin, CH 2 C1-CH(OH)-CH 2 C1, or a,^-dichloro-
hydrin, CH 2 (OH)-CHC1-CH 2 C1, is a colorless liquid insoluble in water and
freely soluble in alcohol and ether. It has an odor like chloroform and a burning,
sweetish taste.
NON-VOLATILE POISONS 93
4. Schindelmeiser's Test. J If nicotine that is not resinous
is treated first with a drop of formaldehyde solution free from
formic acid and then with a drop of concentrated sulphuric acid,
the mixture takes on an intense rose-red color. If nicotine and
formaldehyde are in contact for several hours, the solid residue
obtained gives even a finer color reaction with a drop of nitric
acid. Only a little formaldehyde should be used, otherwise
the solution becomes green after a while and decomposition
takes place.
Under the same conditions, trimethylamine, piperidine, pyridine, picoline,
quinoline and aniline gave no color. Nor did extracts from putrefying horse-
flesh and the entrails of animals, poisoned by arsenic or mercury, give the test,
at least not when these extracts were prepared according to the Stas-Otto method.
5. Physiological Test. When very small quantities of nico-
tine are present, the physiological test should accompany the
chemical tests. A very characteristic picture is given by frogs
after administration of small doses of nicotine. First there is
stimulation, then paralysis of the brain and respiratory muscles
and apparent curare-action (tetanic convulsions). The toxic
action of pure nicotine should be studied first. The experiment
with a frog's heart, which shows temporary cessation of diastole,
is also very characteristic.
ANILINE
Aniline, C 6 H 5 .NH 2 , upon evaporation of the ether extract
from the alkaline solution, will usually appear as reddish or
brownish globules. Dissolve some of this residue in water and
apply the aniline tests already described on page 45. A
further test for aniline consists in mixing some of the residue
with a few drops of concentrated sulphuric acid, and adding a
few drops of potassium dichromate solution. If aniline is
present, an evanescent blue color will appear.
VERATRINE
Pure officinal veratrine is an intimate mixture of two isomeric alkaloids having
the composition Cs2H 49 NO9. These are cevadine, also called crystallized vera-
1 Pharmazeutische Zentral-Halle. 40, 703 (1899).
94 DETECTION OF POISONS
trine, which is nearly insoluble in water; and amorphous veratridine which is
soluble in water. Even small quantities of the crystalline alkaloid will render
veratridine insoluble in water. On the other hand, veratridine will prevent
cevadine from crystallizing. Consequently the crystalline base cannot be iso-
lated by recrystallizing officinal veratrine from ethyl alcohol or from any other
solvent; nor can the water-soluble alkaloid be obtained by simple extraction
with water.
Separation of Cevadine and Veratridine. E. Schmidt uses the following
method to isolate the crystalline and the water-soluble veratrine from officinal
veratrine. Place the officinal preparation in a beaker and dissolve in strong
ethyl alcohol. Heat this solution to 60-70 and add enough warm water to pro-
duce a permanent turbidity. Cautiously add just enough ethyl alcohol to clear
the solution and allow evaporation to take place slowly at 60-70. A white,
crystalline precipitate will presently appear. Filter with suction, wash the pre-
cipitate with a little dilute ethyl alcohol and recrystallize from hot ethyl alcohol.
This is crystalline veratrine. Clear the filtrate from the crystalline precipitate
by adding a little ethyl alcohol and evaporate at 60-70. This will give a,
second crop of crystals. By repeating this process several times one may
obtain in a crystalline condition about one-third of the veratrine taken.
Finally evaporate the filtrate from the crystalline deposit at the given
temperature until there is no longer any odor of ethyl .alcohol. A consider-
able quantity of a resinous mass which is a mixture of both alkaloids will
separate. The aqueous filtrate from this deposit will contain veratridine which
may be obtained by rapidly evaporating the solution in vacua over sulphuric acid.
Properties of Officinal Veratrine. Veratrine appears as a white, amorphous
powder which is crystalline under the microscope. It has a sharp, burning taste
and the minutest quantity introduced into the nostrils excites protracted sneez-
ing. It is almost insoluble in boiling water and the aqueous extract always has a
f aintly alkaline reaction; fairly soluble in ether ( i : 10), benzene, petroleum ether
and amyl alcohol; and freely soluble in ethyl alcohol (i : 4) and chloroform (1:2).
All these solutions have a strong alkaline reaction. Officinal veratrine melts at
1 So-i 55 to a yellowish liquid which solidifies to a transparent, resinous mass.
If the veratrine solution is faintly acid, ether will extract a very little of the alka-
loid. Under the same conditions, chloroform and amyl alcohol will extract
more. The alkaloid is usually deposited from ether as a white, amorphous pow-
der. Phospho-molybdic acid, iodo-potassium iodide, tannic acid and potassium
mercuric iodide give distinct precipitates with an aqueous veratrine solution
containing hydrochloric acid and diluted i : 5000. Chlorides of gold and plati-
num and picric acid fail to show the alkaloid in this dilution.
Constitution. Heated with saturated barium hydroxide, or alcoholic potas-
sium hydroxide solution, crystallized veratrine (cevadine) is hydrolyzed into
angelic acid and cevine:
C,2H4 9 N0 9 + H 2 = CsHsOz 1 + C 27 H 43 NO 8
Oevadme Angelic Cevine
acid
1 Angelic acid (I) and tiglic acid (II) are stereo-isomers :
I. CHr-C-H II. H-C-CHa
CH 3 -C-COOH CH 3 C COOH
NON-VOLATILE POISONS 95
M. Freund 1 has shown that cevadine takes up only one acetyl or benzoyl
group, whereas cevine takes up two. The following formulae show these
relationships:
/O.C 6 H 7 /OH
C 27 H 41 N0 6 < - C 2 7H 41 N0 6 <
X OH \OH
Cevadine Cevine
I i
/O.CO.CH 3
C 27 H 4 ,N0 6 < C 2 7H 41 N0 6 <
X O.CO.CH 3 X O.CO.CH 3
Acetyl-cevadine Diacetyl-cevine
By means of hydrogen peroxide M. Freund has converted cevine into cevine
oxide, C 2 7HNO 9 , which crystallizes well and contains one more atom of oxygen.
V
This compound must belong to the class of the amino-oxides, R = N = O, for
sulphurous acid easily converts it into cevine.
Detection of Veratrine
1. Concentrated Sulphuric Acid Test. Pour a few drops of
concentrated sulphuric acid upon a trace of veratrine. The
alkaloid will have an intense yellow color and, if stirred, will
give a solution of the same color. Gradually, this color will
change to orange, then to blood-red and finally to cherry-red.
Gentle heating will hasten this color change and veratrine, dis-
solved in concentrated sulphuric acid, will give a fine cherry-red
solution almost immediately.
Frohde's and Erdmann's reagents give color changes similar
to those caused by sulphuric acid.
2. Concentrated Hydrochloric Acid Test. If a trace of vera-
trine is dissolved in i or 2 cc. of cold concentrated hydrochloric
acid, the solution will be colorless. When this solution is
heated 10 to 15 minutes in a boiling water-bath, a cherry-red
color will appear. This color will last for a day and even 0.2
mg. of veratrine will produce it.
3. Concentrated Nitric Test. Concentrated nitric acid
dissolves veratrine with a yellow color.
4. Weppen's Test. Thoroughly mix in a mortar i part of
veratrine with about 5 parts of finely powdered cane-sugar.
Add a few drops of concentrated sulphuric acid to some of
1 Berichte der Deutschen chemischen Gesellschaft 37, 1946 (1904). i
96 DETECTION OP POISONS
the mixture upon a watch-glass. At first a yellow color will
appear and later, beginning at the margin this will change to
grass-green and finally to blue. Breathing upon the mixture
will cause the color to change more quickly. Too great an
excess of cane-sugar must be avoided.
E. Laves 1 substitutes an aqueous furfural solution for cane-
sugar in this test. Mix in a test-tube 3 or 4 drops of i per cent,
aqueous furfural solution with i cc. of concentrated sulphuric
acid. Add 3 to 5 drops of this solution to the substance to be
tested so that it just touches the edge of the liquid. If veratrine
is present, a dark streak will gradually run from the substance
into the liquid. At the starting-point it will appear blue or
blue-violet and farther away green. If substance and liquid are
stirred with a glass rod, the liquid will become dark green. After
some time, or more quickly when warmed, the color will become
blue and finally violet.
5. Grandeau's Test. Direct addition of 1-2 drops of bro-
mine water to the yellow solution of veratrine in concentrated
sulphuric acid produces an immediate purple color almost iden-
tical with that appearing when the solution of the alkaloid in
concentrated sulphuric acid stands a long time or is gently
warmed.
6. Vitali's Test. Dissolve veratrine in a few drops of fuming
nitric acid and evaporate the solution to dryness upon the
water-bath in a porcelain dish. A yellowish residue will remain.
If this is cooled and then moistened with an alcoholic potas-
sium hydroxide solution, the color will change to orange-red
or red-violet and stirring will produce a solution having the same
color.
Atropine, hyoscyamine, scopolamine, as well as strychnine,
respond to this test in a very similar manner.
STRYCHNINE
Strychnine, C2iH22N.2O2, occurs with brucine chiefly in nux vomica and
Ignatius beans, constituting the larger part of the mixed alkaloids. The former
contains 2.93-3.14 per cent, of these two alkaloids and the latter 3.11-3.22 per
1 Pharmaceutische Zeitung, 37, 338.
NON-VOLATILE POISONS 97
4
cent. The free base strychnine forms colorless, shining prisms belonging to the
rhombic system which melt at 268. The alkaloid dissolves in 6600 parts of
cold and 2500 parts of hot water, giving alkaline solutions having a very bitter
taste. It is nearly insoluble in absolute ethyl alcohol and in absolute ether. It
dissolves in 160 parts of cold and 12 parts of boiling ethyl alcohol (go per cent, by
volume>; it is also soluble in commercial ether and in benzene; but most readily
in chloroform (6 parts at 15). Strychnine diluted with water 1:600,000 can
be recognized by its bitter taste.
Strychnine is a monacid base combining with one equivalent of acid and form-
ing salts which are usually crystalline. These salts have a very bitter taste and
are very poisonous. The best known strychnine salt, and one used medicinally,
is the nitrate, C2iH22N2O2.HNO3. The combination of one molecule of strych-
nine with one molecule of an alkyl haloid, for example, methyl iodide, to form
strychnine iodo-methylate, C2jH22N 2 O 2 .CH3.I, shows that ' the alkaloid is a
tertiary base. Sodium methylate (CH 3 .ONa) in alcoholic solution converts
strychnine into strychnic acid which is probably an imino-carboxylic acid.
Strychnic acid loses a molecule of water, when its solutions are boiled in presence
of mineral acids, and is changed to strychnine. Because of this behavior Tafel
regards strychnine as an inner anhydride of sfrychnic acid, one containing a
group of the character of an acid imide :
N
Strychnine Strichnic acid
On the basis of Tafel's strychnine formula, strychnine iodo-methylate would be
expressed as follows:
CH 3
(C2oH 22 0)-CO
H
Physiological Action. Strychnine increases reflex irritability of the brain and
spinal cord. Even the slightest stimulus, especially if acoustic, optical, or tactile,
may cause powerful reflexes after large doses of this alkaloid. Convulsions
may follow each stimulus, if the dose is sufficient. Very large doses of strychnine
cause curare-like paralysis of the peripheral ends of motor nerves in frogs and
other warm-blooded animals. It may also affect the muscles of the heart.
Strychnine diminishes the motile power of leucocytes and then arrests their
motion. The poison also affects plant protoplasm, at least that of Mimosa
pudica, in that the plant's motor organs lose their elasticity and flexibility.
Aside from the saliva, bile and milk, the urine is the main channel through
which strychnine is eliminated from the organism. Human urine may contain
even the unaltered alkaloid. Elimination begins during the first hour, is slight
after 2 days but is not complete until much later. More unaltered strychnine is
eliminated after large than after small doses. In the former case 70-75 per cent.
of the alkaloid may remain undecomposed. The liver, kidneys, brain and spinal
cord may store up unchanged strychnine. (See R. Robert, "Intoxikationen.")
7
98 DETECTION OF POISONS
Detection of Strychnine
Sodium and potassium hydroxide, ammonia and alkaline
carbonates precipitate the free base strychnine from aqueous
solutions of its salts as a white crystalline solid:
C 2 iH 22 N 2 2 .HN03 + NaOH = C 2 iH 22 N 2 O 2 + H 2 O + NaNO 3 .
Ether will extract strychnine from an alkaline solution and
deposit the alkaloid on evaporation in fine crystalline needles.
Chloroform takes up the alkaloid more freely, since strychnine
is considerably more soluble in this solvent than in ether. Even
very dilute solutions of strychnine salts give precipitates with
most of the alkaloidal reagents. Tannic acid, potassium mer-
curic iodide and phospho-tungstic acid produce white precipi-
tates; gold chloride and phospho-molybdic acid yellow precipi-
tates; and iodo-potassium iodide brown precipitates. To
obtain tests with these reagents, the residue from ether should
first be dissolved in very dilute hydrochloric acid.
Concentrated sulphuric acid, Erdmann's and Frohde's
reagents dissolve perfectly pure, brucine-free strychnine without
color.
Strychnine is soluble in concentrated nitric acid with a yellow-
ish color. Potassium dichromate, added to solutions of strychnine
salts, precipitates strychnine dichromate, (C 2 iH 22 N 2 O 2 ) 2 .-
H 2 Cr 2 O 7 , in the form of fine yellow crystalline needles which
upon recrystallization from hot water appear as shining orange-
yellow needles.
Potassium ferricyanide, added' to solutions of strychnine
salts, precipitates golden-yellow, crystalline strychnine ferri-
cyanide (C 21 H 22 N 2 O 2 ) 2 .H 3 Fe(CN) 6 + 6H 2 O.
Special Reactions
I. Sulphuric Acid-Dichrcmate Test. Dissolve a very little
strychnine in 2 or 3 drops of concentrated sulphuric acid
upon a watch-glass. The solution should be colorless. Add a
fragment of potassium dichromate and hold it firmly in one
place upon the glass. Intense blue or blue-violet streaks will
come from the potassium dichromate, if the watch-glass is
tilted up and down. If the entire mixture is stirred, the sul-
NON-VOLATILE POISONS 99
phuric acid will have a beautiful evanescent blue or blue- violet
color.
This test may also be made by scattering upon the surface of
the solution of strychnine in concentrated sulphuric acid a few
particles of coarsely powdered potassium dichromate and mix-
ing well with a glass rod. In this way the blue to blue- violet
color reaction is given very beautifully. The blue color is not
permanent. It soon changes to red and finally to dirty green. 1
Strychnine chromate and ferricyanide give this test especially
well. To prepare the former salt, pour a very dilute potassium
dichromate solution over strychnine upon a watch-glass. When
the two substances have been in contact for some time, pour
the remaining liquid from the strychnine chromate. Wash
the precipitate once with a little water. Put some strychnine
chromate upon the end of a glass rod and draw it through a
few drops of concentrated sulphuric acid upon .a watch-glass.
This will produce violet and blue streaks in the acid.
Mandelin's reagent, 2 that is to say, vanadic-sulphuric acid,
gives this strychnine test very well. The blue or violet color
given by this reagent with strychnine is more permanent than
that produced by potassium dichromate. The color finally
changes to orange-red.
Other oxidizing agents may be substituted for potassium dichromate, as
potassium permanganate, lead peroxide, manganese dioxide, 'potassium ferri-
cyanide (see above), cerium oxide and vanadic acid (Mandelin's reagent). But
neither potassium nitrate nor nitric acid can be used, as these reagents even pre-
vent this test. Consequently strychnine nitrate does not give the test.
2. Physiological Test. Dissolve the ether residue in a little
very dilute hydrochloric acid. Evaporate the filtered solution
to dryness upon the water-bath. Dissolve the residue in pure
water (about i cc.) and inject this solution into the lymph-sac
on the back of a lively frog. Keep the experimental frog in a
large, loosely covered beaker. Toxic symptoms will appear in
5 to 30 minutes, depending upon the quantity of strychnine.
According to Tafel (Annalen der Chemie und Pharmazie, 268, 233 (1892),
this color reaction is characteristic of many anilides and is due to the presence
of the group -CO-N =.
2 See page 321 for the preparation of this reagent.
100 DETECTION OF POISONS
Strychnine does not increase reflex irritability for all kinds of
stimuli but only for tactile, optical and especially for acoustic
stimuli. When the dose of strychnine is sufficiently large,
each kind of stimulus mentioned will produce convulsions like
those caused by tetanus. For example, if the beaker contain-
ing the "strychnine frog" is gently tapped, this slight acoustic
stimulus is sufficient to produce convulsions.
Detection of Strychnine in Presence of Brucine
More than traces of brucine prevent detection of strychnine
with concentrated sulphuric acid and potassium dichromate.
Under certain conditions Mandelin's reagent will show strych-
nine more or less distinctly in presence of brucine. Dissolve
the ether residue in concentrated sulphuric acid, if brucine is
present, and add a trace of concentrated nitric acid. A red
color indicates brucine. When the color has changed to yellow,
add a fragment of potassium dichromate and stir. The mixture
will become blue or reddish violet, if strychnine is present.
Solid potassium permanganate, stirred with concentrated sulphuric acid alone,
will give a dark green solution which is yellowish green in a thin layer and assumes
with time a red to violet color on the margin.
The same procedure used to estimate these two alkaloids
quantitatively will permit detection of strychnine even in pres-
ence of considerable brucine. Dissolve the residue containing
brucine in about 2 cc. of dilute sulphuric acid, add 2 drops of
concentrated nitric acid and let the mixture stand 4 hours.
Render alkaline with excess of sodium hydroxide solution and
extract thoroughly with ether. The residue from ether will be
brucine-free or nearly so. Strychnine thus treated will give
very satisfactory tests with concentrated sulphuric acid and
potassium dichromate and with Mandelin's reagent.
BRUCINE
Brucine, Cj3H26N2O4, crystallizes in transparent, monoclinic prisms or shining
leaflets. Crystals from water contain either 4 or 2 molecules and from ethyl alcohol
2 molecules of water of hydration. ' It melts in its water of hydration only a few
degrees above 100, whereas the anhydrous base melts at 178. Brucine is
more readily soluble than strychnine both in water and in ethyl alcohol and there-
fore remains dissolved in the mother-liquors from the preparation of strychnine.
NON-VOLATILE POISONS 101
It is also more soluble than strychnine in ether. Brucine solutions have a very
bitter taste and a strong alkaline reaction. Benzene, but especially chloroform
and amyl alcohol, are excellent solvents for brucine. Brucine differs from strych-
nine in being deposited usually amorphous by evaporation of its ether solution.
Brucine is a monacid, tertiary base and as such forms addition-products with
one molecule of an alkyl iodide. For example, with methyl iodide it gives
brucine iodo-methylate, C23H 2 6NO4.N.CH 3 L With one equivalant of acid
brucine gives in part crystalline salts. Brucine nitrate, C 23 H 2 N 2 O4.HNO3.
2H 2 O, crystallizes in rectangular prisms.
Brucine may be shown by Zeisel's method 1 to contain two methoxyl groups
(-OCH 3 ).
Heated in sealed tube to 80 with sodium and ethyl alcohol, until solution is
complete, brucine is converted into brucic acid, CasH^s^Os.HzO, which contains
an imino-group ( = NH) in its molecule since it forms a nitrosamine. Taf el and
Moufang 2 express the relationship between brucine and brucic acid as follows:
N N
C 2 oH 2 o(OCH 3 )20 CO + H 2 CMC 2 oH 2 o(OCH 3 ) 2 O COOH
\l \
N NH
Brucine Brucic acid
Heated with water, brucic acid is converted into brucine. Consequently
brucic acid is related to brucine as strychnic acid is to strychnine.
Detection of Brucine
Ether, benzene or chloroform will extract brucine from an
alkaline solution. Evaporation of the ether extract usually
leaves the alkaloid in an amorphous condition. The sensitive-
ness of the alkaloidal reagents toward brucine is as follows :
lodo-potassium iodide (i 150,000), Potassium bismuthous iodide (i : 5000),
Potassium mercuric iodide (i 130,000), Phospho-molybdic acid (i : 2000),
Gold chloride (i : 20,000), Tannic acid (i : 2000),
Platinic chloride (i : 1000).
1 Many alkaloids contain one or more, sometimes three or more, methoxyl
groups ( OCH 3 ) united with a benzene nucleus. The determination of the
number of such groups in the molecule is of the greatest importance as a step in
establishing the constitution of an alkaloid, because in this way some of the car
bon, oxygen and hydrogen atoms are at once disposed of. The method employed
for this purpose depends on the fact that all substances containing methoxyl
groups are decomposed by hydriodic acid, yielding methyl iodide and a hydroxyl
compound. By estimating the methyl iodide obtained from a given quantity
of a compound of known molecular weight, it is easy therefore to determine the
number of methoxyl groups in the molecule. This method was first applied by
Zeisel and is of general application. (Perkin and Kipping, "Organic Chem-
istry," page 498.)
2 Annalen der Chemie und Pharmazie 304, 28 (1899).
102 DETECTION OF POISONS
1. Nitric -Acid-Stannous Chloride Test. Concentrated nitric
acid dissolves brucine and its salts with a blood-red color. This
color, however, is slightly stable and soon changes to yellowish
red and finally, especially with heat, to yellow. Add a few
drops of freshly prepared, dilute stannous chloride solution
to this yellowish red or yellow solution. An intense violet
color will appear. Heat usually changes this violet color again
to yellowish red, but addition of a few more drops of stannous
chloride solution will cause the violet color to reappear. The
smaller the quantity of nitric acid, the more likelihood that this
test will give a good result. Colorless ammonium sulphide
solution may be substituted for stannous chloride.
2. R. Mauch's Modification of Nitric Acid-Stannous Chloride
Test. An excellent result can be obtained with this test in the
following manner. Dissolve brucine in 60 per cent, aqueous
chloral hydrate solution and put about 0.5 cc. of this solution
into a test-tube. Add very little dilute nitric acid and thor-
oughly mix the two solutions. Add this mixture to 3 times
its volume of concentrated sulphuric acid so that the former is
on the surface. A yellowish red to deep red zone, depending
upon the quantity of brucine, will appear immediately. When
the upper layer becomes yellow introduce by a pipette a little
stannous chloride solution 1 as a top layer. A brilliant, intensely
violet zone will appear between the two upper layers. The
intensity of this color will gradually increase, especially if the
test-tube is gently tilted to and fro.
ATROPINE
H 2 C CH CH 2 CH 2 OH
N.CH, CH O.CO CH
H 2 C-CH CH, CHs
Atropine, Ci 7 H 23 NO s , crystallizes in shining pointed needles which melt at
115 and dissolve in 600 parts of water, -50 parts of ether and 3.5 parts of chloro-
form. It is also soluble in ethyl alcohol, amyl alcohol and benzene. The aqueous
solution of the alkaloid is alkaline and has a lasting, unpleasant, bitter taste.
Unlike the optically active hyoscyamine, atropine is inactive.
1 Prepare stannous chloride solution by dissolving i part of stannous chloride
m 9 parts of hydrochloric acid having a specific gravity of 1.12 (about 24 per
cent. HC1).
NON-VOLATILE POISONS 103
Constitution. Heated with hydrochloric acid at 120-130. atropine is decom-
posed into tropic acid and tropine:
HOH
H H
H 2 C C CH 2 | CH 2 OH H 2 C C CH 2
| H 3 C N HC O| C CH = H 3 C N HC OH +
H 2 C C CH 2 I O C 6 H 5 H 2 C-
H
Atropine
CH 2 OH
HO C CH
II I
O C 6 H 5
Tropic acid
Heated with barium hydroxide solution, atropine yields atropic acid which is
unsaturated and differs from tropic acid by one molecule of water:
CH 2 .OH CH 2
CH.C 6 H 5 - H 2 O = C.C 6 H 5
I I
COOH COOH
Tropic acid Atropic acid
Since the structure both of tropine and tropic acid has been determined by
synthesis as well as by decomposition, that of atropine is also known. Nitro-
gen in atropine is in the tertiary condition. Hyoscyamine is the stereo-isomer of
atropine. The former, heated at 110 out of contact with air, or allowed merely
to stand in alcoholic solution with addition of a few drops of an alkaline hydroxide
solution, is changed to inactive atropine. . Atropine most likely is the racemic
form, whereas hyoscyamine is the laevo-rotatory modification of this isomeric
base. The degree of rotation of hyoscyamine is [a]o = 20.97. Toward
alkaloidal reagents and when heated with concentrated sulphuric acid, hy-
oscyamine behaves like atropine. It also resembles the latter in giving Vitali's
reaction (see below).
Putrefaction. Ipsen 1 has found atropine very resistant in presence of putre-
fying material. Even after 2 years he could detect the alkaloid which had been
exposed to the influences of decomposition. He experimented with 0.05 gram
of atropine sulphate in respectively 300 cc. of blood, urine and beer and with pure
atropine in 300 cc. of blood.
Detection of Atropine
Ether, benzene or chloroform will extract atropine from a
solution alkaline with sodium hydroxide or carbonate solution.
In a special search for atropine use sodium carbonate solution
and extract with chloroform which is a better solvent than ether.
1 Vierteljahrsschrift fur gerichtliche Medizin und offentliches Sanitatswesen,
3i, 38.
104 DETECTION OF POISONS
Evaporate the solvent and test the residue, which is usually
amorphous, as follows :
1. Vitali's Test. Dissolve the alkaloid in a few drops of
fuming nitric acid, and evaporate the solution in a porcelain
dish to dryness upon the water-bath. Moisten the yellowish
residue when cold with a few drops of a solution of potassium
hydroxide in absolute ethyl alcohol. An evanescent violet
color will appear, if atropine is present.
Hyoscyamine and scopolamine also give Vitali's test. Strychnine and vera-
trine behave similarly. This test therefore is characteristic of the atropine
alkaloids only in the absence of the two latter alkaloids.
2. Para-Dimethylamino-Benzaldehyde Test. 1 The required
reagent is prepared by dissolving 2 grams of para-dimethyl-
CHO (i)
amino-benzaldehyde, CeH^ , in 6 grams of con-
X N(CH 3 ) 2 (4)
centrated sulphuric acid and carefully adding 0.4 gram of
water. The dark yellow solution obtained keeps well for two
weeks. Place a trace of atropine on a watch-glass, add i drop
of the reagent and warm gently. A very intense red-violet
color is produced. This is an exceedingly delicate test.
Hyoscyamine and scopolamine also give this test. The color in the cold with
morphine and codeine is a clear red; with quinine red; with physostigmine and
veratrine green; and with narcotine and papaverine orange.
3. Odor Test. Heat a little atropine in a dry test-tube until
a white vapor appears. An agreeable odor will arise at the same
time. Then add about i cc. of concentrated sulphuric acid, and
heat until the acid begins to darken. Dilute at once with
about 2 cc. of water. During the foaming there will be an in-
tense, sweetish odor like that of honey. By this test, which
was formerly the only method of identifying* atropine, o.oi
gram of the alkaloid can be detected.
4. Physiological Test. Atropine acts in a very characteristic
manner upon the pupil of the eye, and this behavior can be
employed as a test. One drop of an atropine solution diluted
i : 130,000 will produce a noticeable enlargement of the pupil.
1 Chemical Abstracts n, 1518 (1917).
NON-VOLATILE POISONS 105
Dissolve a small portion of the ether residue in 4 or 5 drops of
very dilute sulphuric acid, and introduce a drop of this solution
into a dog's or a cat's eye. The enlargement of the pupil often
persists for several hours. The utmost care should be taken
in performing this test, if applied to the human eye.
The following alkaloidal reagents are especially sensitive
toward atropine: iodo-potassium iodide, phospho-molybdic
acid (i : 10,000), gold chloride, phospho-tungstic acid, potas-
sium mercuric iodide, potassium bismuthous iodide. Picric
acid, added to solutions of atropine salts that are not too
dilute, will precipitate atropine picrate as yellow leaflets.
Platinic chloride gives monoclinic prisms.
HOMATROPINE
Homatropine, CieH^iNOs, is the tropyl ester of phenyl-glycolic or mandelic
acid. The hydrochloride of this base is obtained by heating a mixture of tropine,
jj _ jj _ jj jj mandelic acid and hydrochloric acid, the latter
i| | | acting as a dehydrating agent.
N.CH 3 CH.OOC CH The hydrobromide of homatropine (Ci 6 H 2 i-
I I I NOs.HBr) is used in medicine as a substitute
for atropine. Its action on the pupil is nearly
as strong as that of the natural alkaloid and its effect disappears in 12-24 hours,
whereas that of atropine often lasts 8 days. Moreover it is less toxic than
atropine. Homatropine is a strong tertiary base which forms neutral salts with
acids. This alkaloid does not give Vitali's test. It melts at 92-96; hyoscya-
mine at 108; and atropine at 115.5.
Officinal homatropine hydrobromide may be distinguished from the hydro-
bromides of atropine and hyoscyamine by warming the substance in a test-tube
with a little chloroform. This solvent dissolves the latter two salts in every pro-
portion but homatropine hydrobromide is insoluble. An alternative procedure
consists in dissolving the. given salt in a little water, precipitating the base with
sodium carbonate solution, and extracting with ether. Dehydrate the ether
extract with potassium carbonate and evaporate slowly in a moderately warm
place. This method will give crystals of the alkaloid. Dry these crystals in
aacuo over concentrated sulphuric acid and determine their melting-point.
COCAINE
Cocaine, Ci7H 2 iNO 4 , crystallizes from ethyl alcohol in large, colorless, mono-
clinic prisms which melt at 98. It has a bitterish taste and, placed upon the
tongue, causes temporary, local anaesthesia.
ii.ii
The alkaloid is only slightly soluble in water
N.CH 3 CH OOC C 6 H 5 (i : 700), but easily soluble in ethyl alcohol,
I I ether, chloroform, benzene and acetic ether.
" H2 Its solutions are strongly alkaline and laevo-
rotatory. Dilute acids easily dissolve cocaine and in most cases form readily
106 DETECTION OP POISONS
crystallizable salts. The fixed alkalies, ammonia and alkaline carbonates precipi-
tate the free base from solutions of its salts.
Constitution. Cocaine is a monacid, tertiary base, since it adds a molecule of
CH 3 I. On distillation with barium hydroxide, this alkaloid loses methyl amine
(CHs.NH 2 ), thus proving the attachment of a methyl group to nitrogen. Co-
caine must therefore contain the group = N CHs. This base is also the
methyl ester of an acid and at the same time the benzoyl derivative of an alcoho,!
for it is decomposed into benzoyl-ecgonine and methyl alcohol when heated with
water. If mineral acids, barium hydroxide or alkalies are used instead of water,
the primary product, benzoyl-ecgonine, is further decomposed into ecgonine,
benzoic acid and methyl alcohol. Taking the structural formula proposed by
Willstatter, we may express this reaction as follows :
Ecgonine Bengnc Methyl
H 2 C CH 2 C 6 H 5 CH 3
I CH I
CO O
HC N CH - | H
I H I O
I H
H 2 C C CH + +
O CO
H O
H
Ecgonine (I) heated with phosphorus oxychloride loses a molecule of water and
passes into anhydro-ecgonine (II). The latter heated to 280 with fuming
hydrochloric acid loses carbon dioxide and is converted into tropidine (III).
Tropidine heated with a caustic alkaline solution adds a molecule of water and
passes into tropine (IV), the basic cleavage-product of atropine. Evaporation of
tropine with tropic acid in dilute hydrochloric acid solution yields atropine (V).
Thus it is possible to start with the alkaloid cocaine and synthesize the alkaloid
atropine. The series of changes involved is as follows:'
H.C-CH CH.COOH H 2 C-CH CH.;COOj H
N.CH, CHjOHJ - H 2 N.CH 3 CH - CO 2
:-CH CHJH :! ' H 2 C-CH CH
Ecgonine (I) Anhydro-ecgonine (II)
H 2 C
H,C
H S C-CH CH 2 H 2 C-CH CH 2 CH 2 .OH
CH +H 2 o N.CH, CH.O|H + Hbioc CH:
"Y '
NON-VOLATILE POISONS 107
H 2 C CH CH 2 CH 2 .OH
III I
N.CH 3 CH.O.CO.CH + H 2 O.
H 2 C CH Cn 2
Atropine (V)
CH 2 C 6 H B
Behavior in the Animal Organism. Experiments upon dogs and rabbits show
that the former animal eliminates through the kidneys not more than 5 per cent,
of the cocaine as such and the latter none at all. As the urine of these animals
also contains no ecgonine, the supposition is that the alkaloid is profoundly
changed in the animal organism. The same is true of the human organism.
Proells 1 was able to detect cocaine in cadaveric material at most after 14 days.
In the living organism the alkaloid is said to be changed rapidly into ecgonine.
Detection of Cocaine
Ether, chloroform or benzene will extract cocaine from an
alkaline aqueous solution. Most of the alkaloidal reagents will
precipitate cocaine even from very dilute solutions of its salts.
The reagents especially sensitive are: iodo-potassium iodide,
phospho-molybdic and phospho-tungstic acids, potassium mer-
curic iodide, potassium bismuthous iodide, gold and platinum
chlorides, and picric acid.
Pure concentrated sulphuric and nitric acids, as well as Erd-
mann's, Froehde's and Mandelin's reagents, dissolve cocaine
without color.
i. Precipitation Test. If i or 2 drops of potassium hydroxide
solution are added to an aqueous solution of a cocaine salt not
too dilute, it will become milky. First, resinous globules and
later fine, crystalline needles of the free base, cocaine (melting-
point 98), separate from solution:
Ci 7 H 21 N0 4 .HCl 2 + KOH = dyHziNO, + H 2 O + KC1.
In applying this test to the ether residue, dissolve a consider-
able quantity in a few drops of dilute hydrochloric acid and add
potassium hydroxide solution drop by drop until alkaline and
1 Apotheker-Zeitung 16, 779, 788.
2 Cocaine hydrochloride crystallizes from a concentrated aqueous solution in
fine prisms containing 2 molecules of water which are easily given off. This salt
crystallized from ethyl alcohol is anhydrous and has the formula CnH^NOi.HCl.
The anhydrous compound is the officinal salt.
108 DETECTION OF POISONS
cool well by setting in ice. Special care must be taken to
have the alkaloid pure enough when dry for a melting-point
determination.
This test is not characteristic of cocaine (except the melting-
point which, however, requires considerable pure material),
because most of the alkaloids are precipitated by potassium
hydroxide solution in much the same way.
2. Potassium Pemanganate Test. Add saturated potassium
permanganate solution drop by drop to a concentrated aqueous
solution of a cocaine salt. This reagent will give a violet,
crystalline precipitate of cocaine permanganate. In applying
this test to the ether residue, dissolve a considerable quantity
in 2 drops of dilute hydrochloric acid and evaporate the solution
upon the water-bath. Dissolve the residue in as little water
as possible and add potassium permanganate solution.
3. Chromic Acid Test. Add a few drops of a 5 per cent,
chromic acid solution, or potassium dichromate solution of
corresponding concentration (7.5 per cent.) to a solution of a
cocaine salt. Each drop will produce a precipitate which will
immediately disappear if the solution is shaken. Then add
to the clear solution about i cc. of concentrated hydrochloric
acid which will produce an orange-yellow precipitate more or
less crystalline.
4. Detection of Benzoyl Group. This test requires at least
0.2 gram of cocaine. First, digest the cocaine a few minutes
in a test-tube with 2 cc. of concentrated sulphuric acid upon a
boiling water-bath. Cool and dilute with a little water, all
the while keeping the mixture cold. A white crystalline pre-
cipitate of benzoic acid will appear. Collect and dry this
precipitate upon a filter. Benzoic acid may be recognized by
subliming the precipitate, or, if the quantity is sufficient, by
determining the melting-point (120).
Benzoic acid may also be extracted with ether. Mix the
residue, obtained by evaporating the solvent, with i cc. of
absolute ethyl alcohol and the same quantity of concentrated
sulphuric acid. The characteristic odor of ethyl benzoate,
C 6 H 6 .CO.OC 2 H5, will be recognized.
NON-VOLATILE POISONS 109
5. Reichard's 1 Test. Addition of a concentrated aqueous
solution of sodium nitroprusside, Na 2 Fe(CN) 5 NO.2H 2 O, drop
by drop to a cocaine salt solution, containing at least 4 mg.
of cocaine per cc., causes an immediate turbidity which appears
under the microscope as well-formed reddish crystals. These
crystals will dissolve, if the liquid is warmed, and appear again
if the solution is well-cooled. Morphine does not give this
test.
6. Deniges 2 Test. If an equal volume of 5 per cent, sodium
perchlorate (NaC10 4 ) solution is added to 0.5 per cent, solution
of a cocaine salt, a precipitate consisting of very fine, long needles
is produced. This precipitation may be observed under the
microscope, when the quantity of material is very small.
7. Pisani's 3 Test. Heat cocaine or its hydrochloride witji a
few drops of concentrated sulphuric acid containing 2 per cent,
of formamide (H.CO.NH 2 ). A wine-red color, increasing in
intensity as the temperature rises, is produced. This color
soon disappears and a brownish gray precipitate remains. This
test will detect o.ooi gram of cocaine.
Atropine, quinine, cinchonine, brucine, strychnine, morphine, apomorphine,
codeine and narcotine do not give this test. Papaverine gives a wine-red colora-
tion which changes to yellow, reddish brown and orange.
8. Physiological Test. Dissolve the material (the residue
from the ether extract) in a few drops of dilute hydrochloric
acid and evaporate the solution to dryness upon the water-
bath. Dissolve the residue in a little pure water and apply
this solution to the tongue. Cocaine produces a temporary
anesthesia.
R. Robert ("Intoxikationen") has found small frogs suffi-
ciently sensitive for use in the physiological test for cocaine.
The effects to be observed are dilatation and fixedness of the
pupil, enlargement of the palpebral fissure and also stimulation
1 C. Reichard, Chemiker-Zeitung 28, 299 (1904). Pharmazeutische Zeitung
1904, Nr. 29. Pharmazeutische Zentralhalle 45, 645 (1904).
2 Chemical Abstracts 9, 234 (1915).
3 Chemical Abstracts 9, 511 (1915).
HO DETECTION OF POISONS
of the nervous system. Administer the same quantity of co-
caine hydrochloride to animals for comparison.
PHYSOSTIGMINE
Physostigmine, CuHziNsC^, also called eserine, occurs in the Calabar bean,
the seed of Physostigma venenosum. This alkaloid is deposited from benzene
solution upon spontaneous evaporation of the solvent in large, apparently
rhombic crystals melting at 105. Though but slightly soluble in water, it
dissolves freely in ethyl alcohol, ether, benzene or chloroform. Physostigmine
solutions are strongly alkaline, almost tasteless and laevo-rotatory. It is a strong
monacid tertiary base, forming salts with acids that easily undergo decomposition
and crystallize with difficulty. Light and heat cause acid and alkaline solutions
of this alkaloid to turn red. Owing to this tendency of physostigmine to undergo
decomposition, care must be taken during its isolation to keep it from light and air
and also to avoid rise of temperature. Exclusion as far as possible of free min-
eral acids and caustic alkalies is also desirable.
Detection of Physostigmine
Concentrated sulphuric and nitric acids dissolve physostigmine with a yellow
color which soon changes to olive-green. The alkaloid evaporated upon the
water-bath with fuming nitric acid leaves a residue having a green margin.
Water, ethyl alcohol and sulphuric acid dissolve this residue with a green color.
1. Ammonia Test. If a small quantity of a physostigmine salt is evaporated
to dryness upon the water-bath with ammonium hydroxide solution, a blue or
blue-green residue will remain. This will dissolve in ethyl alcohol with a blue
color. Excess of dilute mineral acid, or acetic acid added to this solution will
change the color to red. The solution is also strongly fluorescent. Examined
spectroscopically, the blue alkaline solution shows one absorption-band in the
red; and the red acid solution one absorption-band in the yellow.
A drop of concentrated sulphuric acid, added to the blue residue from evapora-
tion with ammonia, will give a green solution. The green color diluted with
ethyl alcohol will change to red. If the alcohol is evaporated, the green color
will reappear.
2. Rubreserine Test. If an aqueous solution of a physostigmine salt is shaken
for some time with an excess of potassium or sodium hydroxide solution, a red
coloring-matter, rubreserine (CisHnNjC^), is formed. This compound separates
as red needles which become greenish blue on further oxidation owing to forma-
tion of eserine blue.
Barium hydroxide solution may be substituted for the caustic alkali. This
reagent first produces a white precipitate which soon becomes red on being
shaken. Sometimes this change occurs even in the cold but invariably takes
place with heat.
3. Physiological Test. The marked action of physostigmine in causing con-
traction of the pupil is very characteristic. It is advisable to use the cat's eye
for this test. Even o.i mg. of this alkaloid will produce noticeable contraction.
NON-VOLATILE POISONS 111
CODEINE
Codeine, Ci 7 Hi 8 (CH 3 )N03, the methyl ether of morphine, crystallizes from
water, or from ether containing water, in colorless, transparent octahedrons
jj which are often very large. These crystals are quite
H H 2 | easily soluble in water. One part of the free base
C C N is soluble at 15 in 80 parts of water and at 100 in
^\/\/\ 15 parts. Codeine differs from most of the other
I || I I 2 alkaloids, morphine, for example, in its relatively high
CH 3 O.C C C CH 2 solubility in water. Ethyl alcohol, ether, amyl alcohol,
\/\^\/ chloroform and benzene also dissolve codeine freely.
C C CH j t j S; however, practically insoluble in petroleum
O-HC CH ether. Aqueous codeine solutions are strongly alka-
\/ line and bitter. Pure codeine does not reduce iodic
C acid, nor does it immediately produce a blue color or
_/ X-VTT a blue precipitate in a mixture of potassium ferri-
cyanide and ferric chloride solutions. A pure
codeine solution is also not colored blue by ferric chloride solution alone. (Dif-
ference between morphine and codeine.) Phospho- molybdic acid, iodo-
potassium iodide, potassium bismuthous iodide and potassium mercuric iodide
give precipitates even with very dilute codeine solutions. On the other hand,
tannic and picric acids, gold and platinum chlorides are less sensitive.
Detection of Codeine
1. Sulphuric Acid Test. Concentrated sulphuric acid dis-
solves codeine without color. After long contact or upon appli-
cation of gentle heat, the solution will have a reddish to bluish
violet color. The solution of codeine in concentrated sulphuric
acid, heated to about 150 and then cooled, is colored deep red
by a drop of concentrated nitric acid.
2. Nitric Acid Test. Cold nitric acid (25 per cent.) will con-
vert codeine into nitro-codeine (Ci 8 H 2 o(NO2)NO 3 ). At the
same time the acid will dissolve the alkaloid with a yellow color
which soon changes to red. Concentrated nitric acid dissolves
codeine with a reddish brown color.
3. Oxidation Test. Mix a little codeine upon a watch-glass
with four times the quantity of finely powdered potassium arsen-
ate (KH 2 AsO 4 ). Add a few drops of concentrated sulphuric
acid and then warm gently over a small flame. The acid will
have a deep blue or blue- violet color, if the codeine is not quite
pure. Excess of potassium arsenate does not affect the test.
112 DETECTION OF POISONS
If water or sodium hydroxide solution is added, the blue color
will change to orange-yellow.
A trace of ferric chloride solution may be substituted for potassium arsenate.
Sulphuric acid containing i drop of ferric chloride solution to 10 cc. of acid is
prescribed by the German Pharmacopceia for detecting the alkaloid in codeine
phosphate.
4. Froehde's Test. This reagent dissolves codeine with a
yellowish color which soon changes to green and finally to blue.
Gentle warming of the solution over a very small flame will
hasten this change of color.
R. Mauch warms 2 or 3 drops of a chloral hydrate solution of codeine with i
drop of Froehde's reagent. An intense blue color finally appears.
5. Formalin-Sulphuric Acid Test. 1 Concentrated sulphuric
acid containing formalin dissolves codeine with a reddish
violet color which changes to blue-violet. This color is per-
sistent. The spectrum shows an absorption of orange and
yellow.
6. Furfural Test. 2 Dissolve codeine in a few drops of con-
centrated sulphuric acid and warm very gently with a drop of
cane-sugar solution which must not be in excess. This will
produce a purple-red color.
This test may also be made by mixing a drop of sugar solution
with codeine, dissolved in about 5 drops of 50-60 per cent, aque-
ous chloral hydrate solution, and then adding 1-2 cc. of con-
centrated sulphuric acid as an under layer. A carmine-red
ring will appear at the zone of contact. The color is quite
permanent and increases in intensity upon standing. If the
sulphuric acid and 'chloral hydrate solution are thoroughly
mixed, the entire liquid will be red. After a time the shade of
color will be more of a red-brown.
7. Pellagri's Test. Both codeine and morphine give this
test. Dissolve codeine in concentrated hydrochloric acid and
1 See preparation of reagents, page 321.
2 This test depends upon furfural formed by the action of concentrated sul-
phuric acid upon cane-sugar. Very dilute aqueous furfural solution (i: 1000)
may be substituted for cane-sugar. Excess of furfural unlike cane-sugar does
not interfere with the test. Tr.
NON-VOLATILE POISONS 113
add at the same time 3-4 drops of concentrated sulphuric acid.
Expel hydrochloric acid upon the water-bath and heat the resi-
due about 15 minutes. Dissolve the dirty red or violet residue
in 2-3 cc. of water, add a few drops of hydrochloric acid and
neutralize with acid sodium carbonate. Then add alcoholic
solution of iodine drop by drop (2 to 4 drops) and shake thor-
oughly for several minutes. An emerald-green solution indi-
cates codeine. Extract the green solution with ether. The
color of the ether will be red, whereas that of the aqueous solu-
tion will remain green. This is a test for apomorphine (see
page 128) formed from codeine by the mineral acid.
Ci7H 18 (CH 3 )N0 3 + HC1 = CnHnNOs + CH 3 .C1 + H 2 O.
Codeine Apomorphine
8. Mecke's Test The reagent, consisting of selenious acid
and concentrated sulphuric acid, 1 dissolves codeine with a blue
color quickly changing to emerald-green and finally becoming
a permanent olive-green.
NARCOTINE
Xarcotine, CszH^NO?, crystallizes in shining prisms or in tufts of needles
which are nearly insoluble in cold water but readily soluble in boiling ethyl alcohol
or chloroform. Separation of alkaloid from the cold alcoholic solution is almost
complete. At 15 narcotine dissolves in 170 parts of ether; 31 parts of acetic
ether; and 22 parts of benzene. Solutions of narcotine are not alkaline nor
bitter. In these respects narcotine is very
OCH 3 different from the other opium alkaloids. Salts
I of narcotine do not crystallize, their stability is
^\ slight and their solutions react acid. Salts with
HC C.OCH 3 volatile acids are decomposed, when their solutions
are evaporated, with separation of narcotine. So-
dium acetate precipitates free narcotine from its
C
solution in hydrochloric acid.
Constitution. Narcotine is a monacid, tertiary
HC O base and as such combines with i mol. of CH 3 .I,
CH O C CH forming narcotine methyl iodide (C 22 H 23 NO 7 .-
3 ^\ /\ CH 3 I). This compound is formed at ordinary
/ O C C N.CHs temperatures but the reaction is hastened by
Cx heat. Narcotine heated with hydriodic acid loses
~C ^ CH 2 3 methyl groups wh ich form CH 3 I. The al-
^ kaloid must therefore contain 3 methoxyls,
H H 2 3(CH 3 O-), in the molecule. Heated with water
to 140, with dilute sulphuric acid, or even with
See preparation of reagents, page 322.
114 DETECTION OF POISONS
barium hydroxide solution narcotine is hydrolyzed into nitrogen-free opianic
acid and into the basic and consequently nitrogenous hydrocotarnine:
C 22 H 23 NO 7 + H 2 O = CioH 10 p 6 + Ci 2 Hi 6 N0 3
Narcotine Opianic Hydrocotarnine
acid
By oxidative cleavage, that is, by treatment of narcotine with such oxidizing
agents as nitric acid, manganese dioxide and sulphuric acid, lead dioxide and
ferric chloride, cotarnine and opianic acid are the products :
C 22 H 23 N07 + (H 2 O + O) = CioHiqOs + Ci 2 H 16 NO 4
Narcotine Opianic Cotarnine
acid
Evidently these cleavage-products show that this alkaloid is made up of two
complexes, one nitrogen-free and the other containing nitrogen. The chemical
constitution of these cleavage-products has been determined and is expressed by
the following formula?:
H CH 3
C | H 2
S\ C C
CH 3 C CH
/O C C N CH 3
C-C=0 H 2 C< | || |
H \0 C C CH 2
CH 3 C C C=O H 2 C
\/
C
HO C=O H H 2
Opianic Acid Hydrocotarnine
CH 3 O O
I /"
C C H
-C C NH.CH 3
H 2 C<( | || |
X C C CH 2
YY
H H 2
Cotarnine
On the basis of these results, Roser and Freund have proposed the structural
formula for narcotine given above. They consider the constitution of this alka-
loid as definitely settled. If the formula of narcotine is compared with that of
hydrastine (see page 116), a great similarity in structure will be seen. In fact
narcotine is a methoxylized hydrastine.
Detection of Narcotine
Narcotine is so feebly basic that chloroform will extract the
alkaloid completely from an aqueous tartaric acid solution.
Consequently its separation from the rest of the opium alkaloids
as well as from other alkaloids is easy. Naturally ether or
chloroform will also extract narcotine from an aqueous alkaline
NON-VOLATILE POISONS 115
solution. The alkaloid as it comes from its ether solution is
usually a slightly colored, varnish-like residue which hardens
after a time to a mass of radiating crystals. Narcotine is
precipitated from its hydrochloric or sulphuric acid solution
by iodo-potassium iodide, phospho-molybdic acid, potassium
mercuric iodide, potassium bismuthous iodide even in consider-
able dilution (i : 5000).
1. Sulphuric Acid Test. Dissolved with stirring in concen-
trated sulphuric acid, narcotine produces a greenish yellow color
which gradually changes to reddish yellow and finally after
several days to raspberry-red.
2. Dilute Sulphuric Acid Test. A solution of narcotine in
dilute sulphuric acid (i 15), evaporated on the water-bath in a
porcelain dish or over a very small flame, has a reddish yellow
color, changing with stronger heat to crimson-red. As the acid
begins to^ evaporate, blue- violet streaks radiate from the margin
and finally the entire liquid has a dirty red-violet color (Dragen-
dorff 's reaction) . The same color changes appear, if the yellow-
ish solution of narcotine in concentrated sulphuric acid is
heated very carefully.
3. Froehde's Test. This reagent dissolves narcotine with a
greenish color. If concentrated Froehde's reagent is used, the
green color changes immediately to cherry-red, especially upon
application of gentle heat. This color is quite persistent.
4. Couerbe's Test. Dissolve narcotine in cold concentrated
sulphuric acid and mix a trace of nitric acid with this solution
after 1-2 hours. A red color will appear and gradually become
more and more pronounced.
Erdmann's reagent gives the same color change.
5. Wangerin's Test. 1 Place a mixture of o.oi gram of
narcotine with 20 drops of pure concentrated sulphuric acid
and 1-2 drops of i per cent, cane-sugar solution upon a watch-
glass and heat upon the water-bath with stirring about i minute.
At first the solution has a greenish yellow color which passes
through yellow, brownish yellow, brown and brown-violet into
an intense blue-violet.
1 Pharmazeutische Zeitung, 48, 607 (1903).
116 DETECTION OF POISONS
The intensity of this color increases somewhat upon standing
and the blue-violet color persists several hours.
Applied to apomorphine, atropine, brucine, quinine, codeine, caffeine, hydras-
tine, morphine, physostigmine, pilocarpine and strychnine, this test gives
solutions that are colorless or nearly so. Only the morphine solution after a
while has a pale pink color. Coniine and narcotine have a light yellow color;
narceine chestnut-brown; and picrotoxin salmon color to pale pink.
Colchicin, digitalin and veratrine behave toward this reagent as toward pure
concentrated sulphuric acid without the addition of the small quantity of sugar.
In this test 1-2 drops of i per cent, aqueous furfural solution may be substi-
tuted for the sugar solution. From yellow, brown, olive and other colors there
finally emerges a deep, clear, dark blue. The brilliancy of this color increases
somewhat on standing. After several hours there is a gradual change to a pure
green color. For the detection of traces of narcotine (o.ooi gram) use a i per
cent, sugar solution.
6. Selenious Acid-Sulphuric Acid Test. This reagent dis-
solves narcotine with a greenish steel-blue color which after a
time becomes cherry-red. Heat immediately discharges the
cherry-red color.
HYDRASTINE
Hydrastine, C 21 H 2 iNO 6 , occurs together with berberine, C 2 oH 17 NO 4 , and cana-
dine, CjjoHziNCh, in hydrastis root, the root of Hydrastis canadensis, to the
amount of 1.5 per cent, and more. The fluid ex-
O.CH 3 tract prepared from this root and used in medicine
^ contains 2-2.5 per cent, of hydrastine.
^\ Preparation. Extract hydrastis root with hot
HC C.OCH 3 water containing acetic acid. Filter the solution,
I II evaporate to a thin extract and add 3 vols. of dilute
\/ sulphuric acid (1:5). Nearly all the berberine
C separates out in fine yellow crystals as acid sul-
phate, C 2 oHi7NO4.H 2 SO 4 . Precipitate hydrastine
H C -- from the mother-liquor of berberine sulphate by
JJ means of ammonium hydroxide solution and purify
the alkaloid by crystallization from acetic ether or
p N.CHj ethyl alcohol. Hydrastine crystallizes from ethyl
\O__ p p ptr alcohol in rhombic prisms melting at 132. It is
\/\ / nearly insoluble in water but freely soluble in hot
C C etli yl alcohol, benzene or chloroform. This alkaloid
H H 2 has a bitter taste and its solutions are alkaline.
Hydrastine solutions are optically active. In chloro-
form this alkaloid is laevo-rotatory, whereas in dilute hydrochloric acid it is
dextrorotatory.
C
NON-VOLATILE POISONS 117
Constitution. The constitution of hydrastine is entirely analogous to that of
narcotine (see page 113). On oxidation with dilute nitric acid hydrastine gives
opianic acid and hydrastinine :
C 2 iH 21 N0 6 + (H 2 + O) = C 10 H 10 5 + C u Hi 3 NO 3
Hydrastine Opianic acid Hydrastinine
Hydrastine is a monacid base which is shown to be a tertiary base by its
behavior toward alkyl iodides, for example, with CH 3 I it forms hydrastine methyl
iodide, C2iH2iNO6.CHsI, which crystallizes in needles. Hydrastine contains
two methoxyl groups, because when heated with hydriodic acid according to
Zeisel's method two such groups are removed.
Since the chemical nature of opianic acid has long been known, the only
problem is the explanation of the nature of hydrastinine, the other cleavage-
product. The constitution of hydrastinine, as well as that of many other alka-
loids, has been determined by A. W. Hofmann's method of exhaustive methyla-
tion. 1 Hydrastinine (I) is a secondary base which forms, when heated with an
excess of CHsI, hydrastinine hydriodide and trimethyl-hydrastyl-ammonium
iodide (II). Heated with alkalies, this ammonium iodide is decomposed into
trimethylamine, hydriodic acid and nitrogen-free hydrastal (III). The latter
on oxidation gives hydrastic acid (IV) which was recognized as the methylene
ether of nor-meta-hemipinic acid (V) :
/CH:0 + 2 CH 3 I =
(I) (CH 2 2 )C6H 2 <
X CH 2 .CH 2 .NH.CH 3
/CH:O
(CH 2 2 )C 6 H 2 < V
X CH 2 .CH 2 .N(CH 3 ) 3 I
Hydrastinine Trimethyl-hydrastyl-
annnonium iodide
/CH:0
(II) (CH 2 2 )C 6 H 2 <
X CH 2 .CH 2 .N(CH 3 ) 3 I + KOH =
/CH:O
(CH 2 2 )C 6 H/ + KI + H 2 + N(CH S ) 3
CH:CH 2
Hydrastal
1 When the nitrogen of an organic base becomes quinquevalent, it is more sub-
ject to change. Hofmann (Liebig's Annalen, 78, 263 (1851) showed, for example,
that tetra-ethyl-ammonium hydroxide breaks up on heating into triethylamine,
ethylene and water:
CH 2 C 2 H 5 \
P 2 J} 5 >N-OH =|| + C 2 HAN + H 2 0.
C 2 H 5 2
Nitrogen in alkaloids on treatment with an alkyl haloid (e.g., CH 3 I) combines
with it in many instances, forming compounds having a structure analogous
to that of tetra-ethyl-ammonium hydroxide. This process is called "exhaustive
methylation." Upon decomposition these derivatives yield products which
often throw light upon the structure of the alkaloid.
118 DETECTION OF POISONS
CH-0 /COOH
(III) (CH,0 2 )CH<( ; CH Oxidized = ( CH ') C ' H < COOH
Hydrastic acid
Hydrastic acid and nor-meta-hemipinic acid are identical. The latter has the
structure (V):
H
C
O C C.COOH
< | ||
\0 C C.COOH
(V) H 2 C< | ||
C.
C
H
Nor-meta-hemipinic acid
From these and other relations it has been determined that cotarnine is a
methoxy-hy drastinine :
H CH 3 .0 H
H / II
C C=0 C C=0
/O C C NH.CH, /QC C NH.CH 3
H 'o4 I! k H ' C i!
YY YY
H H 2 H H 2
Hydrastinine Cotarnine
The alkaloid narcotine is a methoxy-hydrastine (see page 144).
Detection of Hydrastine
1. Concentrated Sulphuric Acid dissolves hydras tine without
color but upon being gently warmed the solution becomes violet.
2. Froehde's Reagent dissolves hydrastine with a green
color which gradually changes to brown.
3. Mandelin's Reagent dissolves hydrastine with a rose-
red color which immediately changes to orange-red and gradu-
ally fades.
4. Fluorescence Test. Dissolve hydrastine in dilute sul-
phuric acid, shake vigorously and add drop by drop very
dilute potassium permanganate solution. Hydrastinine is
formed and the solution shows a beautiful blue fluorescence.
The ether extract of the alkaline solution on evaporation
leaves hydrastinine in a crystalline condition.
NON-VOLATILE POISONS 119
QUININE
Quinine, CaoH24N 2 O 2 , is precipitated amorphous and anhydrous from solutions
of its salts by caustic alkalies, alkaline carbonates or ammonia. On standing,
H however, it gradually becomes crystalline, forming a
C hydrate with 3 molecules of water of hydration.
x There are also other hydrates of quinine. Anhy-
_, CH.CH:CH 2 drous quinine melts at 173; the trihydrate at 57.
i 2 An ether solution on evaporation usually deposits
HO.C CH 2 CH 2 tnis alkaloid as a resinous, or varnish-like, amorphous
\ | / residue. Quinine is soluble in about 2000 parts of
cold and 700 parts of boiling water; and freely solu-
-jj ble in ethyl alcohol, ether or chloroform. Solutions of
H quinine in sulphuric, acetic or tartaric acid exhibit a
C beautiful blue fluorescence. In the case of the sul-
==, phate this fluorescence is distinctly visible in a dilu-
MU U L.UL.J13
H/l
J.1
-C,
HC C CH Hydrochloric, hydrobromic and hydriodic acid do
not give fluorescent solutions of quinine. These acids
N C even discharge the fluorescence, if added to a fluor-
escent quinine solution.
Constitution. Quinine is a diacid, ditertiary base, the salts of which with
i and 2 equivalents of acid are usually crystalline. The salts with i equivalent
of acid are the more stable. Quinine hydrochloride, C 20 H 24 N 2 O 2 .HCJ.2H 2 O,
used in medicine, crystallizes in long delicate tufts of needles. The diter-
tiary character of quinine is shown by the fact that it unites with 2 mole-
cules of methyl iodide, for example, to form quinine dimethyliodide,
C 2 oH 2 4N2O2.2CH3l. Quinine must contain an hydroxyl group, since it can
form a mono-benzoyl and a mon-acetyl-quinine. Moreover one methoxyl
group has been found in the quinine molecule. The difference empirically
between cinchonine, Ci9H 2 2N 2 O, and quinine, C2oH24N2O 2 , is CH 2 O. Every
investigation of these substances has shown that quinine is a methoxy-cincho-
nine. For example, on oxidation with chromic acid, cinchonine gives cinchonic
acid which was recognized as quinoline -y-carboxylic acid; whereas quinine under
the same conditions gives quinic acid, or p-methoxy-cinchonic acid:
COOH(-y) COOH( 7 )
HI' H
C C C C
HC 7 C X CH (p)CH 3 O.C / C \H
ni c in ^
H H
Cinchonic acid Quinic acid
Both alkaloids on oxidation also give the nitrogenous compounds mero-
quinene, cincholoiponic acid and loiponic acid. Consequently there is no
doubt that cinchonine and quinine contain two nitrogenous nuclei, one of which
120 DETECTION OF POISONS
is a quinoline complex. The second nucleus is connected with the latter in the
7-position, as the formation of cinchonic and quinic acids shows. Meroquinene,
cincholoiponic acid and loiponic acid, derived by oxidation with chromic acid
from the so-called "second half" of the cinchonine and quinine molecules, form
a continuous series of oxidation products, since meroquinene can be oxidized to
cincholoiponic acid and the latter to loiponic acid. The following formulae best
explain the chemical behavior of these three compounds:
CH 2 .COOH
CH 2 .COOH
COOH
A H
CH
C \ H '
H 2 C CH CH :
: CH 2 H 2 C CH.COOH
H 2 C CH.COOH
H 2 C CH 2
H 2 C CH 2
H 2 C CH 2
\/
\/
\/
N
N
N
H
H
H
Meroquinene
Cincholoiponic acid
Loiponic acid
The structural formula already given for quinine was propose^ by W. Koenigs 1
and is based on the results of his own experiments as well as on those of \V. V.
Miller and of Skraup. Cinchonine has hydrogen in place of the methoxyl group
in the quinoline nucleus; otherwise the two alkaloids are identical in structure.
Detection of Quinine
Ether, benzene or chloroform will extract quinine from an
aqueous alkaline solution. Ether on evaporation deposits the
alkaloid as a resinous, amorphous varnish in which its presence
may be recognized by the following tests:
1. Fluorescence Test. Dissolve the residue from the ether
extraction of the alkaline solution in a little dilute sulphuric
acid. If quinine is present, this solution will exhibit blue
fluorescence.
2. Thalleioquin Test Dissolve quinine in a few drops of
very dilute acetic acid and add 5-10 drops of saturated chlorine
water. The colorless solution has a faint, blue fluorescence.
Excess of ammonium hydroxide solution will produce an emer-
ald-green color. A solution containing considerable quinine
will give a green precipitate. This precipitate (thalleiioquin)
is always an amorphous substance, the composition of which
has not been determined. It is soluble in ethyl alcohol and
chloroform but not in ether.
1 Meroquinene and the Structure of the Cinchona Alkaloids; Annalen der
Chemie und Pharmazie 347, 147 (1906^.
NON-VOLATILE POISONS 121
E. Polacci recommends the following procedure for the thal-
le'ioquin test. Gradually heat quinine (about o.oi gram) to
boiling with a little lead dioxide (PbO 2 ) , 2-3 cc. of water and 2
drops of dilute sulphuric acid. Let the solution settle and
either decant or filter. Finally, carefully add 5-6 drops of
ammonium hydroxide solution as a top layer. A beautiful
green ring will appear at the zone of contact.
Interferences with the Thalleioquin Test. Antipyrine interferes with this test.
Mixtures of i per cent, solutions of antipyrine and quinine give finally a beautiful
red instead of a green color. This interference does not cease until these two
substances are in the proportion of 0.25 parts of antipyrine to 5 parts of quinine.
Caffeine also interferes with the thalleioquin test, when the proportion is 2 parts
of quinine to 3 parts of caffeine. Other compounds like urea prevent the appear-
ance of this color, whereas morphine, pilocarpine, cocaine, atropine, codeine,
strychnine, carbolic acid and chloral hydrate have no effect upon the thalleioquin
test.
H. Fiihner 1 has shown that the thalleioquin reaction is connected with the
p-oxyquinoline complex. Chlorine passed into a solution of pure p-oxy-quino-
line cooled with ice produces a white crystalline precipitate. This substance
crystallizes from petroleum ether in colorless prisms or tabular crystals melting
at 58. Structurally it is 5,5-dichloro-6-keto-quinoline. Solutions of this di-
chloro-keto-quinoline and of its hydrochloride are colored a pure green or blue
by ammonium hydroxide. Fiihner thinks 5,6-quinoline quinone is probably
formed and gives the green color with ammonia.
H H
C C
H Cl,
C C
H
C C
J^\. y/\
HC C C.OH(p)
HC C CH
HC C CO
1 II 1 -
HC C CH
HC C CO
HC C CH
N C
H
p-Oxy-quinoline
N C
H
5,5-Dichloro-keto-
quinoline
N C
H
5,6-Quinoline
quinone
3. Herapathite Test. Mix 30 drops of acetic acid, 20 drops
of absolute ethyl alcohol and i drop of dilute sulphuric acid.
Add 20 drops of this mixture to o.oi gram of quinine and heat
to boiling. Finally add i drop of an alcoholic solution of iodine
(i : 10) or 2 drops of o.i n-iodine solution. When the solution
has stood for some time, green leaflets with a metallic luster will
1 Berichte der Deutschen chemischen Gesellschaft 38, 2713 (1905).
122 DETECTION OF POISONS
form. This is an iodine compound of quinine called "Hera-
pathite," having the constant composition
This substance can be recrystallized from boiling ethyl alcohol.
Herapathite crystals are pale olive-green by transmitted light
but by reflected light they have a beautiful, cantharidin-
green, metallic luster.
Caustic alkalies, ammonia, sulphurous acid and hydrogen sulphide decompose
herapathite. A. Christensen recommends keeping on hand the following reagent
for the herapathite test:
Parts
Iodine i
Hydriodic acid (50%; i
Sulphuric acid 0.8
Ethyl alcohol (70%) 50
Add a few drops of this reagent to the alcoholic solution to be tested for quinine.
4. Hirschsohn's Test. 1 If i drop each of 2 per cent, hydro-
gen dioxide and 10 per cent, copper sulphate solution are added
to a neutral solution of quinine hydrochloride or sulphate at
boiling temperature, a more or less intense raspberry-red color
will appear. This color soon passes through blue-violet into
blue and after a time into green. A quinine solution (i :
10,000) will still give a distinct red- violet color.
Excess of acid as well as of ethyl alcohol interferes with this test. The
behavior of a solution of aloes toward this test is similar to that of quinine.
Of the alkaloidal reagents potassium bismuthous iodide is
especially recommended as a precipitant of quinine. With
quinine sulphate solutions this reagent produces precipitates
having an intense yellowish red color. Shaken with sodium
hydroxide solution this precipitate is decomposed and unaltered
quinine can be obtained by extraction with ether and evapora-
tion of the ether solution. H. Thorns 2 has made use of this
reaction in the quantitative separation of quinine from mixtures.
CAFFEINE
Since caffeine (see page 84) is a weak base, ether will extract
only a little of the alkaloid from the tartaric acid solution. The
1 Pharmazeutische Zentral-Halle 43, 367 (1902).
2 Berichte der Deutschen pharmazeutischen Gesellschaft 16, 130 (1906).
NON-VOLATILE POISONS 123
greater part will be in the ether extract of the alkaline solution.
Ether usually deposits caffeine in white, shining needles ar-
ranged in clusters. Caffeine dissolves in ether with some
difficulty and the alkaline solution should be extracted several
times. For the tests characteristic of this alkaloid see page 85.
ANTIPYRINE
Most of the antipyrine (see page 82) is obtained by extract-
ing the alkaline solution with ether. It is usually purer from
the acid than from the alkaline solution and frequently appears
in crystalline leaflets. Antipyrine differs from most alkaloids
in having only a faintly bitter taste and in being freely soluble
in water. To identify antipyrine, dissolve the ether residue in a
little water and divide the solution into two equal parts. Test
one portion with ferric chloride solution and the other with
fuming nitric acid.
Detection of Antipyrine in Urine. The color of urine after administration of
antipyrine is intensely yellow to blood-red. Part of the antipyrine in the organ-
ism appears in the urine as oxy-antipyrine-glycuronic acid and another part is
unchanged and can usually be detected directly in urine by ferric chloride solu-
tion. A safer procedure is to add excess of ammonia to a considerable quantity of
urine and extract with chloroform. Evaporate the solvent, dissolve the residue
in a little water and test the filtered solution for antipyrine with ferric chloride
solution and with fuming nitric acid.
Antipyrine is easily absorbed. The urine may show a reddish color, even an
hour after the drug has been taken, and give a test with ferric chloride solution.
The red color disappears in about 24 hours but the elimination of antipyrine is
not complete in that time. Its detection is still possible after 36 hours. A con-
venient procedure is to add to the urine as an upper layer very dilute ferric chlo-
ride solution. A red ring will appear if the urine contains antipyrine. Jonescu 1
states that antipyrine in the human organism passes unchanged into the urine.
Only a small portion and large doses of the drug must have been taken is
eliminated in conjugation with sulphuric acid. Conjugation with glycuronic
acid 2 (see above) according to Jonescu does not occur in the human organism.
1 Berichte der Deutschen pharmazeutischen Gesellschaft 16, 133 (1906).
H\
2 Glycuronic acid, C 6 Hi O: = ^C(CH.OH) 4 COOH, may be regarded as a
?
derivative of glucose. Possibly it occurs in normal urine in small quantity as
a conjugated acid. After administration of various alcohols, aldehydes, ketones,
phenols (chloral hydrate, camphor, phenol, thymol, menthol, borneol), there
takes place in the animal organism often after oxidation or reduction a con-
jugation of these substances with glycuronic acid.
124 DETECTION OF POISONS
PYRAMIDONE
Pyramidone, or 4-dimethyl-amino-antipyrine, Ci 3 Hi 7 N 3 O, has been exten-
sively used in medicine of late as an antip>retic and anodyne. It is a white,
crystalline powder, nearly tasteless and readily soluble
in water. It melts at 108. Its aqueous solution has
jsj a neutral reaction. Ether removes only traces of
pyramidone from acid solution, but extracts it easily
CH 3 N 2 sCO an d completely from alkaline solution. Ether usually
H _Qs = 4(i N(CH 3 ) 2 de P sits this substance in fine needles. Pyramidone
is also freely soluble in ethyl alcohol, ether, chloroform
or benzene. It is a strong reducing agent and in this respect differs from
antipyrine. For example, pyramidone will reduce gold chloride even in the
cold, whereas antipyrine and tolypyrine require heat.
Preparation. Antipyrine dissolved in concentrated acetic acid is converted by
treatment with potassium nitrite into nitroso-antipyrine which appears as green
crystals. This compound dissolved in ethyl alcohol may be reduced by zinc and
acetic acid to amino-antipyrine. The latter, dissolved in methyl alcohol and
treated with methyl iodide and potassium hydroxide, is converted into dimethyl-
amino-antipyrine, or pyramidone.
C 6 H 5 C 6 H 6 C 6 H 6
I I I
N N N
CH 3 .N CO CH 3 .N CO ' CH 3 .N CO + 2 CH 3 I
- | | +4H-+ | -
CH 3 .C=C|H HOj.NO CH 3 .C=C.NO CH 3 .C=C.NH 2 2 KOH
Antipyrine Nitroso-antipyrine Amino-antipyrine
C 6 H 5
N ' * /
CH 3 .N CO
CH 3 .C=C.N(CH 3 ) 2
Pyramidone
Behavior in the Organism. Human urine, if neutral or faintly acid, usually
has a bright purplish red color after administration of pyramidone. After stand-
ing for some time it will deposit a sediment consisting of red needles soluble in
ether or chloroform but especially in acetic ether. Jaffe 1 recognized this com-
pound as rubazonic acid, a pyrazolone derivative. Isolation of rubazonic acid
from urine may be brought about as follows. Acidify fresh urine with hydro-
chloric acid and let it stand in an open dish. The acid will appear as small red.
particles. Ferric chloride solution produces a blue- violet color in the acid liquid
filtered from rubazonic acid. This filtrate contains most of the product formed
from pyramidone in animal metabolism, namely, crystalline antipyryl-urea
melting at about 245.
^erichte der Deutschen chemischen Gesellschaft 34, 2737 (1901); and 35,
2891 (1902).
NON-VOLATILE POISONS 125
C 6 H 8
A
CH 3 .N CO
CH 3 .C=C.NH.CO.NH 2
Antipyryl-urea
Detection of Pyramidone
1. Ferric Chloride Test. Ferric chloride solution added to
pyramidone produces a blue-violet color which soon changes to
reddish violet and then disappears.
2. Fuming Nitric Acid Test. A few drops of fuming nitric
acid, added to a solution containing pyramidone, give a blue to
blue-violet color.
3. Bromine Water Test. This reagent imparts a grayish
color to pyramidone solutions. With concentrated solutions
it produces an inky color.
4. Iodine Test. Tincture of iodine colors an aqueous pyra-
midone solution blue.
5. Guglialmelli's 1 Test. Either of the following two reagents
may be used, but (6) usually gives the better result:
(a) Arseno-tungstic Solution. Dissolve 25 grams of sodium tungstate
(Na 2 WO4.2H 2 O) in 200 cc. of cold distilled water, adding 20 grams of pure
arsenic trioxide (As2Os) and boiling the solution for 1.5 hours under a reflux-con-
denser. Filter the resulting light, bluish green solution when cold and bring the
volume to 250 cc.
(b) Arseno-tungsto-molybdic Solution. Boil in the same manner 10 grams
of sodium tungstate, 2 grams of sodium molybdate (Na2MoO4-ioH2O) and 10
grams of pure arsenic trioxide for 1-2 hours with 75 cc. of distilled water. Bring
the cold solution to a volume of 100 cc.
Added to aqueous pyramidone solutions, these reagents
produce white spots. Those from reagent (a) are soluble in
alkali with an intense blue color; whereas those from reagent
(b} give an intense indigo color. Pyramidone produces these
colors in a dilution of i : 750,000.
These reagents produce white spots with antipyrine solutions but they are
soluble in alkali without color.
1 Chemical Abstracts 12, 664 (1918).
126 DETECTION OF POISONS
6. Palet's 1 Test. A few drops of a freshly prepared solution
of potassium ferricyanide and ferric chloride, added to an
aqueous pyramidone solution, produce the characteristic blue
color and precipitate of Prussian blue. The reaction is very
sensitive. Morphine gives the same test (see page 135).
With antipyrine the reagent gives a blood-red color and precipitate and is
negative with phenacetine, acetanilide and caffeine. If pyramidone and anti-
pyrine are together, a little hydrochloric acid should first be added.
C. Extraction of the Ammoniacal Solution with Ether and Chloroform
(a) Ether Extract. Apomorphine and traces of morphine. 2
(/3) Chloroform Extract. Morphine and narceine. (It may
also contain antipyrine and caffeine. 3 )
The aqueous alkaline solution (see page 86) , separated from
ether, must be tested further for the substances under a and 0.
Apomorphine may be recognized by the green color of the
aqueous acid solution. Excess of sodium hydroxide solution
causes oxidation, especially if the solution is exposed for any
length of time to air, and gradually changes the color to deep
purple-red. Moreover, the ether extracts, both of the acid
and alkaline solutions, are red or violet-red when apomorphine
is present. Solutions, examined by the Stas-Otto method, not
having these characteristics, need not be tested for apomorphine.
In that case proceed at once with the morphine and narceine
tests.
To extract apomorphine, morphine and narceine with the
proper solvent, the aqueous solution separated from ether, which
is alkaline from sodium hydroxide solution (see page 86), must
be rendered alkaline with ammonium hydroxide solution.
First acidify the solution with dilute hydrochloric acid (test
with blue litmus paper) and then add ammonium hydroxide
solution until alkaline.
1 Chemical Abstracts, 13, 216 (1919).
2 Ether dissolves traces of freshly precipitated, amorphous morphine.
Antipyrine and caffeine, though freely soluble in chloroform, dissolve with
lifficulty in ether. The latter solvent frequently fails to extract these substances
completely from aqueous solution. They will then appear in the chloroform
NON-VOLATILE POISONS 127
(a) If there is any indication of apomorphine, first extract the
ammoniacal solution repeatedly with ether and then several
times with hot chloroform for the morphine and narceine tests.
(/3) If there is no indication of apomorphine, extract the
ammoniacal solution several times direct with hot chloroform
(see below).
APOMORPHINE
Constitution. Apomorphine, CiTHnNOz, is a monacid, tertiary base with two
phenol hydroxyl groups. According to R. Pschorr 1 it has the structural formula
here given.
Properties. Apomorphine is an amorphous base
W (9) readily soluble in ethyl alcohol, ether, benzene or
C C* N 3 chloroform and colored green in contact with air.
,/\/\/\ Aqueous and alcoholic apomorphine solutions, origi-
HC C CH CH 2 nally colorless, soon turn green in the air from oxida-
iwn r r r rw ^ on ' Solutions of apomorphine thus changed by
' >\ /v xx s 2 oxidation are emerald-green. Ether and benzene solu-
C C C(8) tions are purplish violet; those in chloroform blue-
1 || | violet. Being phenolic in character, apomorphine
(4) HO HC CH resembles morphine in its solubility in sodium hy-
0^ droxide solution. Alkaline solutions of the alkaloid
jj absorb oxygen from the air and become brown or
even black in color. Apomorphine differs from mor-
phine in being more soluble in water and in ethyl alcohol, but especially in
being soluble in ether, benzene and cold chloroform, in which morphine is
almost insoluble.
Formation and Preparation. Sulphuric, hydrochloric, phos-
phoric and oxalic acids, the alkalies and zinc chloride have
mainly a dehydrating action upon morphine and convert it
into apomorphine:
C, 7 Hi9N0 3 = H 2 + CirHuNO*
Morphine Apomorphine
Codeine, the methyl ether of morphine, also gives apomor-
phine when heated at 140 with concentrated hydrochloric acid.
l8 2 3 HCl = H 2 O + CH 3 C1 + CirHnNOi
Codeine Apomorphine
Apomorphine is prepared by heating morphine (i part) with
concentrated hydrochloric acid (20 parts) for 3 hours in an
autoclave at 130-150.
1 Berichte der Deutschen chemischen Gesellschaft 39, 3124 (1906); and 40,
1984 (1907).
128 DETECTION OF POISONS
(a) Detection of Apomorphine in the Ether Extract
Ether will not extract apomorphine from a solution contain-
ing tartaric acid but will dissolve its colored oxidation products.
This solvent behaves similarly toward solutions of this alkaloid
in sodium or potassium hydroxide solutions. Ether or chloro-
form will extract apomorphine only from a solution alkaline
with ammonium hydroxide. Ether solutions of apomorphine
usually deposit a greenish residue. A characteristic of this
alkaloid is its strong reducing action. For example, it will re-
duce iodic acid with liberation of iodine and produce a purple
color with gold chloride. Apomorphine gives the following
tests:
1. Sulphuric and Nitric Acids. Concentrated sulphuric acid
dissolves apomorphine without color. Addition of a drop of
concentrated nitric acid to such a solution produces an eva-
nescent violet color that soon changes to blood-red and finally to
yellowish red. With concentrated nitric acid alone this alka-
loid gives a violet-red color that soon becomes red-brown and
finally brownish red.
2. Pellagri's Test. Dissolve apomorphine in dilute hydro-
chloric or sulphuric acid and first add acid sodium carbonate in
excess. Then add drop by drop 1-3 drops of an alcoholic iodine
solution and shake for several minutes. The solution will have
a blue-green or emerald-green color. Extract with a little ether
and the solvent will become violet, whereas the aqueous
solution will remain green.
3. Froehde's Test. This reagent dissolves pure apomorphine
with a green color. If the alkaloid has been acted upon by air
to any extent, the color is violet.
4- Wangerin's 1 Test. L Prepare a fresh solution of apomor-
phine hydrochloride (about i per cent.). Add 4 drops of potas-
sium dichromate solution (0.3 per cent.) to i cc. of this solution
and shake for about i minute. The solution will have an in-
tense dark green color. Then add 10 cc. of acetic ether and
shake again. This solvent will become violet. Finally add
1 Pharmazeutische Zeitung 47, 599 and 739-740 (1902).
NON-VOLATILE POISONS 129
from a pipette about 5 drops of stannous chloride solution 1
(i per cent.) and shake well. The color of the acetic ether layer
will change to green and, upon further addition of a few drops of
potassium dichromate solution, the acetic ether will again be-
come violet. If 10 cc. of chloroform are substituted for acetic
ether in this test, the oxidation product of apomorphine will im-
part the same violet color to the chloroform. But if stannous
chloride solution is added carefully, the color will change to pure
indigo-blue and persist upon further agitation with potassium
dichromate solution.
'5. E. Schmidt's Tests. 2 (a) A drop of very dilute ferric
chloride solution (i : 100) will color 10 cc. of an aqueous apo-
morphine hydrochloride solution blue even in a dilution of
i : 10,000.
(&) Shake 10 cc. of the same apomorphine hydrochloride
solution with i cc. of chloroform. Then render alkaline with
sodium hydroxide solution and at once shake with air. The
aqueous solution becomes evanescent violet in color and the
chloroform blue.
6. Palet's Test. 3 Add 1-2 drops of apomorphine solution
to 1-2 cc. of Guglialmelli's reagents. 4 After shaking the
mixture for 2-3 minutes, add 5-10 cc. of a cold, saturated
solution of pure sodium carbonate. An indigo-blue color,
varying in intensity with concentration and time, appears.
Divide the liquid after 5 minutes into 3 portions. These treated
respectively with amyl alcohol, benzene and acetic ether give
rise to an intense blue, dark violet and violet color. Further
addition of 1-2 drops of 10 per cent, stannous chloride solution
to the acetic ether extract changes the violet color to emerald-
green as in Wangerin's test. Apomorphine diluted 1:500,000
gives a distinct blue color, and a slightly positive result when
1 Prepare this reagent as follows :
Crystallized stannous chloride (SnCl 2 .2H 2 O; i gram
Hydrochloric acid (25 per cent.) 50 cc.
Water 50 cc.
2 Apotheker-Zeitung 23, 657 (1908).
3 Chemical Abstracts 12, 601 (1918).
4 See page 125 for the preparation of these reagents.
130 DETECTION OF POISONS
diluted i : 1,000,000. In all cases the intensity of the color
increases on standing.
Morphine gives the same blue color which is not extracted by solvents. Nar-
cotine and narceine do not give this reaction.
(/3) Examination of the Chloroform Extract
Preliminary Morphine Test. As a preliminary test for mor-
phine, acidify a small portion of the aqueous alkaline solution
separated from ether (see page 85) with dilute sulphuric acid,
add iodic acid solution and extract' with a little chloroform. If
the latter has a violet color from dissolved iodine, morphine
may be present. But a final conclusion regarding the presence
of morphine must not be drawn from a positive test, since there
are many other organic substances besides this alkaloid that
will reduce iodic acid. 1 This is a delicate preliminary test for
morphine and that is its only value. If it is negative, morphine
is probably absent.
To detect morphine and narceine positively, render the aque-
ous solution alkaline with ammonium hydroxide and extract at
once as already directed (see page 126) with considerable hot
chloroform 2 in a capacious flask. Separate the two liquids as
usual in a separatory funnel. Several extractions of the aque-
ous solution with fresh portions of hot chloroform are necessary
because of the slight solubility of morphine even in boiling
chloroform. Should the chloroform and the aqueous solution
form a refractory emulsion that will not separate, add a few
drops of ethyl alcohol, set the flask on a warm but not boiling
water-bath and carefully turn the flask from time to time. This
procedure usually causes the immediate separation of the two
liquids. Place the combined chloroform extracts in a dry flask,
add a few crystals of dry sodium chloride or anhydrous sodium
sulphate to remove adherent water, pour the chloroform when
clear through a dry filter and evaporate in not too large a glass
1 In testing animal matter that contained no morphine, the author has repeat-
edly obtained extracts that strongly reduced iodic acid.
2 C. Kippenberger (Zeitschrift fur analytische Chemie 39, 201, 290) uses
chloroform, containing 10 per cent, of ethyl alcohol by volume, to extract
morphine.
NON-VOLATILE POISONS 131
dish placed upon a warm water-bath. The chloroform may also
be filtered directly into the dish as fast as it evaporates. If the
residue is bitter and can be scraped together with a platinum
spatula or a pocket-knife, test for morphine and narceine. 1 In
testing for morphine use Froehde's, Husemann's and Pellagri's
tests as well as those given by formalin-sulphuric acid and iodic
acid. The presence of morphine is not established unless all
these morphine tests give positive results. If the quantity of
the residue from chloroform permits, test for morphine with
ferric chloride solution. This test is very characteristic of
morphine but requires more than traces for a satisfactory result.
Purification of Impure Morphine
When the chloroform residue is too impure, especially if red
or biown, it must be purified. Dissolve in hot amyl alcohol
and shake the solution thoroughly with several portions of hot
water containing a few drops of dilute sulphuric acid. The acid
dissolves the morphine, whereas the amyl alcohol retains most of
the coloring matter. Add ammonium hydroxide solution in
'excess to the acid solution and extract several times with hot
chloroform. The morphine obtained by evaporation of the
chloroform should be nearly pure.
MORPHINE
Morphine, CirHigNOs, crystallizes from dilute ethyl alcohol in shining prisms
which are colorless and transparent and but slightly soluble in water (i: 5000
at 15; and 1:500 at 100). These solution.
CHs are very bitter and have an alkaline reactions
H HZ | Crystalline morphine is insoluble in ether and
x,^ xx xx benzene. The amorphous alkaloid is soluble in
JJQ CH cfj 2 amyl alcohol, hot chloroform and acetic ether.
ill | | Solutions of the hydroxides of ammonia, potassium
C C CHz or sodium and sodium carbonate solution precipi-
^^C ^p rvrr tate free morphine from solutions of morphine
| | | salts.
~~/9 , CH!! Constitution. Morphine is a mon-
H C acid, tertiary base whose nitrogen is in
jj OH union with three atoms of carbon. The
three oxygen atoms have different func-
tions. One is a phenolic hydroxyl and gives to morphine th
1 Antipyrine and caffeine may also be in this residue (see above).
132 DETECTION OF POISONS
character of a monatomic phenol. Conseqeuntly when sodium
hydroxide solution is added drop by drop to a morphine salt
solution, there is first a precipitate of crystalline morphine
(a) which is freely soluble in excess of alkali (0) but is again
precipitated on addition of ammonium chloride solution (7) :
(a) Ci 7 H l8 N0 2 (OH).HCl + NaOH = CnHisNCMOH) + H 2 O + NaCl,
09) Ci 7 Hi 8 N0 2 (OH) + NaOH = CwHwNOrfONa) + H 2 O,
(y) CnHisNCMONa) + (H 4 N)C1 = CnH l8 NO 2 (OH) + NH 5 + NaCl.
Hydrogen of this phenolic hydroxyl may be replaced also by
alkyl groups and acid radicals. In codeine this hydrogen is
replaced by methyl. A second oxygen atom of morphine is
alcoholic and the third is indifferent. The latter like the oxygen
of an ether is combined with two carbon atoms and forms a so-
called bridge-oxygen atom.
Of the 17 carbon atoms of morphine 14 belong to the phenan-
threne nucleus, 1 since the nitrogen-free cleavage-products of
morphine and codeine, namely, morphol and morphenol, have
been identified as phenanthrene derivatives. R. Pschorr has
synthesized morphol which is 3,4-dioxyphenanthrene. Mor-
phenol contains two hydrogen atoms less and may be converted
into morphol by i eduction with nascent hydrogen. These two
phenanthrene derivatives have the following structural formulae :
H H
C C
/\ /\
HC CH HC CH
i L;; t li
H/V H/VX
II I II I o
HC C HC C /
\/\ \S\/
C C.OH( 4 ) C C
(i)HC C.OH( 3 ) HC C.OH
\/ V
(2)C C
H H
Morphol Morphenol
/ \ /CH = CH\
Phenanthrene, Ci 4 H 10) HC^ >C C/ >CH, occurs
C - CH^
in coal-tar together with anthracene. It forms colorless crystals which melt at
99 and boil at 340. It is readily soluble in ether or benzene and with difficulty
in ethyl alcohol. Phenanthrene solutions exhibit bluish fluorescence.
NON-VOLATILE POISONS 133
By distillation over zinc dust morphenol may be reduced to
phenanthrene.
The structural formula of morphine written above was pro-
posed by R. Pschorr 1 and seems to explain most satisfactorily
the reactions of this alkaloid.
Morphine is easily oxidized. This may be brought about in
alkaline solution by atmospheric oxygen. Potassium per-
manganate or ferricyanide and ammoniacal copper solution
may also be used. As a result the non-toxic oxy-dimorphine,
also called pseudomorphine, which is soluble in caustic alkali,
is formed:
2 C 17 Hi9N0 3 + O = (Ci7H 18 NO ? )2 + H 2 O.
Morphine Oxydimorphine
Detection of Morphine
1. Nitric Acid Test. Concentrated nitric acid dissolves mor-
phine with a blood-red color which gradually changes to yellow.
Stannous chloride or ammonium sulphide solution will not re-
store the violet color of a solution that has become yellow.
(Distinction from brucine.)
2. Husemann's Test. Dissolve morphine upon a watch-
glass in a few cc. of concentrated sulphuric acid. The solution
is colorless. Heat for 30 minutes upon the water-bath, or over
a small flame for a very short time until white fumes arise. A
reddish or brownish color appears. Cool and add 1-2 drops of
concentrated nitric acid. A fugitive, reddish violet color ap-
pears and soon changes to blood-red or yellowish red. This
color gradually disappears.
A preferable procedure is to dissolve morphine in cold con-
centrated sulphuric acid and add a trace of concentrated nitric
acid after the solution has stood in a desiccator 24 hours. A
small crystal of potassium nitrate or chlorate may be substi-
tuted for nitric acid.
Frequently impure morphine is obtained from the chloroform extract of a
solution prepared from animal material., Such a residue gives a more or less
1 Berichte der Deutschen chemischen Gesellschaft 40, 1984 (1907).
134 DETECTION OF POISONS
highly colored solution with sulphuric acid. Heat usually intensifies the color.
But even under these conditions it is possible to detect the red color caused by
nitric acid or potassium nitrate.
3. Pellagri's Test. Proceed as described for codeine. (See
page 112.) Avoid excess of alcoholic iodine solution, otherwise
the latter may mask the green color.
4. Froehde's Test. This reagent dissolves morphine with a
violet color which passes through blue to dirty green and finally
to faint red. These colors vanish on addition of water.
5. Formaldehyde-Sulphuric Acid Test. The solution used
for this test is called Marquis' reagent. 1 With a trace of mor-
phine it produces a purple-red color which changes to violet
and finally becomes pure blue. This blue solution, kept in a
test-tube and only slightly exposed to air, retains its color for
some time. Codeine and apo morphine give the same violet
color. Narcotine also gives violet solutions but they become
olive-green and finally yellow. Oxy-dimorphine gives a green
color.
6. lodic Acid Test. Shake a solution of morphine in dilute
sulphuric acid with a few drops of iodic acid and chloroform.
Morphine will liberate iodine which will dissolve in chloroform
with a violet color.
Obviously this delicate test is conclusive for morphine only in the absence of
other reducing substances.
7. Ferric Chloride Test. Add 1-2 drops of neutral ferric
chloride solution to a neutral solution of a morphine salt. A
blue color appears. In testing the chloroform residue, dissolve
in a little very dilute hydrochloric acid. Evaporate this solu-
tion to dryness upon the water-bath, dissolve the residue in pure
water and add a drop of ferric chloride solution.
8. Lloyd's Test. Lloyd has found that a mixture of morphine,
hydrastine and concentrated sulphuric acid alone without
potassium dichromate will produce the same violet color given
by the latter with a solution of strychnine in concentrated sul-
1 Mix 2-3 drops of 40 per cent, formaldehyde solution with 5 cc. of concen-
trated sulphuric acid and use a few drops of this mixture for the morphine test.
NON-VOLATILE POISONS 135
phuric acid. Lloyd's reaction is of value in the detection of mor-
phine or hydrastine only when more than traces of both alka-
loids are present. A. Wangerin 1 considers these reactions
characteristic only when 0.0050.01 gram of morphine and
0.002-0.01 gram of hydrastine are present.
Make an intimate mixture of about these quantities of both
alkaloids upon a watch-glass. Add 5 drops of pure concen-
trated sulphuric acid and stir the mixture for 10 minutes
over a white background. In the center the color-tone is a
clear red- violet and more or less of a blue-violet in the thinner
marginal region. Apomorphine hydrochloride, treated in the
same way with hydrastine and concentrated sulphuric acid,
gives almost the same reaction as morphine.
9. Prussian Blue Test. Add a few drops of a dilute mixture
of ferric chloride and potassium ferricyanide solutions to a
morphine salt solution. A deep blue color appears. Consid-
erable morphine produces a precipitate of Prussian blue.
Potassium ferricyanide oxidizes morphine to oxy-dimorphine :
+ 2KOH + K 6 Fe 2 (CN)i 2 = aH 2 O + (CnHuNO^ + 2K 4 Fe(CN)
Morphine Potassium Gxy-dimorphine Potassium
ferricyanide ferrocyanide
Potassium ferrocyanide then forms Prussian blue with ferric
chloride.
10. Silver Test. Warm a morphine salt solution with silver
nitrate and excess of ammonium hydroxide solution. Mor-
phine produces a gray precipitate of metallic silver.
11. Bismuth Test.^Dissolve morphine in concentrated
sulphuric acid and sprinkle a little bismuth subnitrate on the
surface of the solution. A dark brown color appears.
12. G. Fleury's Test. 2 Dissolve morphine in a little very
dilute sulphuric acid (about 0.05 normal), add some lead di-
oxide (PbO 2 ) and shake for 6-8 minutes. A pale rose color
appears. Addition to the nitrate of ammonium hydroxide
solution in excess produces a brown color which persists for
1 Pharmazeutische Zeitung 46, 57 (1903;.
2 Annales de Chimie analytique appliquee 6, 417 (1907).
136 DETECTION OF POISONS
several hours. When the quantity of substance is very small,
stir on a porcelain color-plate for 6-8 minutes with a drop of
dilute sulphuric acid and a minute particle of lead dioxide.
When the insoluble matter has settled, tilt the porcelain plate
so that the clear solution runs up the side. A drop of am-
monium hydroxide solution now gives a brown color.
13. Dan Radulescu's Test. 1 Add a small particle of sodium
nitrite to a very dilute morphine salt solution, then a dilute
acid and render alkaline with concentrated potassium hydroxide
solution before all the gas has escaped. The solution when con-
centrated has a pale rose to a deep ruby-red color. Acids dis-
charge but alkalies restore this color. This reaction is said to
be characteristic of morphine bases and especially adapted
for the detection of morphine in mixtures.
14. Diazonium Test. 2 - In presence of alkalies, morphine and
its salts form dyestuffs with diazonium compounds. The
reagent is a solution of diazotized sulphanilic acid.
Diazonium Reagent. Dissolve 0.2 gram of sulphanilic acid
/ /NH 2 (i)\
I C 6 H 4 <^
n-HCl and 10 cc. o.i n-NaNOj.
/NH 2 /N = N
C6H ^SO H + HCI + NaN 2 = CeH4 \ '
Dissolve 0.243 gram of morphine sulphate in 100 cc. of water.
Render 5 cc. of this solution alkaline with sodium carbonate or
bicarbonate and add an equal volume of diazonium reagent.
A red color, changing to orange on addition of acid, immediately
appears. The color diluted i : 10,000 is faint red; i : 100,000
distinctly yellow; and i : 2ooo 3 intensely dark red in a thin
layer.
No opium alkaloid except morphine gives this reaction; not even the synthetic
derivatives of this alkaloid (dionine, peronine and heroine). Other alkaloids
1 Chemisches Zentralblatt 1906, i, 1378.
2 Lautenschlaeger: Chemical Abstracts 13, 22^2 (1919).
3 If the above proportions are used and the test is made as directed, the dilu-
tion will be i : 2000.
lin 80 cc. of water, cooling with ice. Then add 10 cc. o.i
NON-VOLATILE POISONS 137
giving colors are: emetine and physostigmine (red); sparteine and piperidine
(yellow); coniine and nicotine (bright yellow). But the morphine color above
is stable in acid solution.
General Alkaloidal Reagents. The reagents of this class
especially sensitive toward solutions of morphine salts are:
lodo-potassium iodide, Potassium bismuthous iodide,
Phospho-tungstic acid, Phospho-molybdic acid,
Potassium mercuric iodide, Gold chloride.
Plantinic chloride after some time causes a granular orange-
yellow precipitate. Tannic acid causes no precipitate, or at
most only a very slight cloudiness which becomes somewhat
more pronounced with time.
Behavior of Morphine in the Animal Organism. The mucous lining of the
stomach, rectum or respiratory passages as well as open wounds absorb mor-
phine. The alkaloid injected hypodermically acts more rapidly and more po-
tently than when absorbed from the stomach. Marquis 1 found that morphine
disappears very quickly from the blood but is firmly retained by certain organs
like the brain. Some absorbed morphine is conjugated with glycuronic acid and
some is oxidized but the rest of the alkaloid is eliminated unchanged. Faust
has found that morphine is transformed or destroyed only in men and animals
habituated to the poison but is eliminated unchanged nearly quantitatively in
the faeces in the case of organisms not immunized. Morphine appears in the
urine only in very small quantity after medicinal doses. In men and dogs a not
insignificant quantity of the morphine taken is eliminated by the glands of the
gastro-intestinal tract, even when the alkaloid has been subcutaneously injected.
Marquis found that more than 30 per cent, of intravenously injected mor-
phine is deposited in the liver in the course of 15 minutes. The alkaloid is
present at first in this organ in the free state and then is soon combined or trans-
formed. The conjugation of morphine in the brain also begins very soon. Free
morphine is also rapidly changed in the blood, spleen, kidneys and in the mucous
lining of the intestines. Marquis states that always in acute and even more so
in chronic morphine poisoning a large quantity of the poison leaves the blood and
is stored in the salivary glands, mucous lining of the stomach and large intestine,
kidneys, spleen, liver and is withdrawn by these organs from the brain and spinal
cord.
Morphine is quite resistant to putrefaction. The author 2 detected this alka-
loid positively in animal material containing morphine (stomach and intestines
together with contents) which had stood for 15 months in a glass vessel and had
completely putrefied in presence of insufficient air. Doepmann 3 obtained the
1 Arbeiten des Dorpater Instituts, ed Robert, 14 (1896).
2 Berichte der Deutschen Pharmazeutischen Gesellschaft n, 494 (1901).
3 Chemical Abstracts 9, 1663 (1915).
138 DETECTION OF POISONS
same result by mixing definite quantities of morphine hydrochloride with lean
horse meat and allowing putrefaction to take place from one to eleven months.
NARCEINE
OCH 3 Narceine, C 23 H 27 N6 8 .3H 2 O, crystallizes from
water or ethyl alcohol in prisms which melt at
/,--. 165 when air-dried. The alkaloid has a faintly
HC C.OCHs bitter taste. Though only slightly soluble in cold
water, it is freely soluble in hot. When a hot
H( ^ C.COOH satura ted aqueous solution of narceine is cooled,
Q it solidifies to a crystalline mass. Narceine is in-
soluble in ether, benzene or petroleum ether and
CO is soluble only with difficulty in cold ethyl alcohol ,
CH 3 O. | amyl alcohol or chloroform. In detecting nar-
A>\ / 2 jj ceine it is important to know that it is not ex-
/O.C C N<^ tracted by ether, benzene or petroleum ether from
H 2 C<(' | || I ^CH 3 a solution rendered alkaline by potassium or
-C , C . CH 2 sodium hydroxide solution. It is, however,
Q extracted by hot chloroform or amyl alcohol
H H 2 from an aqueous solution rendered alkaline by
ammonium hydroxide solution.
Constitution. Narceine is a weak tertiary base in which two
methyl groups are attached to nitrogen. By means of Zeisel's
method it may be shown that the molecule also contains three
methoxyl groups. Narceine, being soluble in caustic alkalies
and forming esters with alcohols, must contain a carboxyl
group. The alkaloid must also contain a carbonyl group (CO),
since it forms a hydrazone with phenyl-hydrazine. The nar-
ceine formula above may therefore be resolved into :
C 23 H 2T NO 8 = CieHuON (CH 3 ) 2 (OCH 3 ) 3 (CO) (COOH).
The narceine molecule contains neither an alcoholic nor a
phenolic hydroxyl group, since it forms no acetyl derivative
with acetic anhydride. There is a close relationship between
narceine and narcotine. By heating narcotine iodo-methylate
with sodium hydroxide solution Roser converted this compound
into a base called pseudo-narceine. Freund has recently shown
that Roser's pseudo-narceine is identical with the opium alka-
loid narceine and explains the conversion of narcotine into nar-
ceine by saying that the iodo-methylate loses i molecule of
hydriodic acid and takes up i molecule of water:
NON-VOLATILE POISONS
139
OCH 3
OCH 3
HC
Hi
C.OCH 3
Ico
CH 3 JHi C O
/ ai 1
/O.C C N CH 3
H 2 C< | || |\CH,
X O.C C CH 2
\/ \S
C C
H H 2
Narcotine iodo-methyolate
+ H 2
-HI
CH 3
H 2 C
HC C.OCHs
I II
HC C.COOH
V/
c
CO
CH 2
CH 3
Y
Cns
H 3
CH 2
H H 2
Narceine
All the reactions and transformations of narceine can easily
be explained on the basis of this structural formula.
Detection of Narceine
1. Sulphuric Acid Test. Concentrated sulphuric acid dis-
solves narceine with a grayish brown color, which gradually
changes to blood-red. This reaction takes place at once with
heat.
2. Dilute Sulphuric Acid Test. Narceine, warmed in a porce-
lain dish upon the water-bath with dilute sulphuric acid until
a certain concentration is reached, gives rise to a fine violet
color which changes after long heating to cherry-red.
3. Froehde's Test. At first a solution of narceine in this
reagent has a brownish green color which gradually changes
to green and finally to red. Gentle heat hastens this reaction.
4. Iodine Test. Aqueous iodine solution (iodine water) or
iodine vapor colors solid narceine blue.
Morphine interferes with or entirely prevents this reaction.
5. Erdmann's Test. This reagent, as well as concentrated
nitric acid, dissolves narceine with a yellow color which heat
changes to dark orange.
6. Chlorine -Ammonia Test. Pour a few drops of chlorine
water upon narceine and add, while stirring, a few drops of
140 DETECTION OF POISONS
ammonium hydroxide solution. A deep red color immediately
appears.
7. Resorcinol-Sulphuric Acid Test. 1 Mix thoroughly upon
a watch-glass resorcinol (o.oi to 0.02 gram) with 10 drops of
pure concentrated sulphuric acid. Add a trace of narceine
(about 0.002 to 0.005 gram) and, while stirring, warm the in-
tensely yellow solution upon a boiling water-bath. A carmine-
red to cherry-red color appears. As the solution cools, this
color begins at the margin to change gradually to more of a
blood-red and finally after several hours to orange-yellow.
8. Tannin-Sulphuric Acid Test. Mix narceine (0.002 to
o.oi gram) with tannin (o.oi to 0.02 gram) and 10 drops of
pure concentrated sulphuric acid. Heat with constant stirring
upon the water-bath and the color of the solution, which is
yellowish brown at first, soon becomes pure green. If heat is
applied for some time, the green color changes to blue-green
and finally through a more or less blue tone to a dirty green.
Tannin-sulphuric acid gives a similar color test with narcotine and hydras-
tine which closely resemble narceine in constitution.
Of the general alkaloidal reagents potassium zinc iodide 2
precipitates narceine even in a dilution of i : 1000. It is a
white, filiform precipitate which after a time becomes blue.
This blue color appears immediately, if a trace of iodine solu-
tion is added to the reagent.
Of the other general reagents iodo-potassium iodide, potassium mercuric
iodide, potassium bismuthous iodide and phospho-molybdic acid are char-
acterized by considerable delicacy toward narceine.
SYNOPSIS OF GROUP II
Stas-Otto Method
A. Ether Extract of Acid Solution may Contain :
Picrotoxin. Very bitter. Reduces Fehling's solution with
heat.
Melzer's test: red streaks radiating from picrotoxin with
alcoholic benzaldehyde + cone. H 2 SO 4 .
1 A. Wangerin, Pharmaceutische Zeitung, 47, 916 (1902).
2 See page 319 for the preparation of this reagent.
NON-VOLATILE POISONS 141
Cone. H 2 S0 4 : soluble with yellow or orange-red color; drop
of K^C^OT + Aq has brown margin.
Langley's test: picro toxin + 3 parts KNO 3 , moistened with
cone. H2SO4, red with excess of saturated NaOH + Aq.
Colchicin. Very bitter. Yellowish and amorphous. Dilute
mineral acids render aqueous solutions intensely yellow.
Cone. HNO 3 : soluble with dirty violet color changing to
brownish red and finally to yellow; excess of KOH + Aq
renders orange-yellow or orange-red.
Zeisel's test: boil yellow colchicin solution in cone. HC1 in test-
tube 2-3 minutes with 2 drops of FeCl 3 + Aq. Green or olive-
green when cold, especially if diluted with equal volume of water.
Picric Acid. Very bitter. Yellow. Material and extracts
more or less intensely yellow.
Isopurpuric acid test: aqueous picric acid, gently warmed
with a few drops of saturated KCN + Aq, gives red color.
Picramic acid test: aqueous picric acid, warmed with few
drops of (H 4 N) 2 S + Aq, becomes red.
Dyeing test: aqueous picric acid dyes wool and silk intense
yellow but not cotton.
Acetanilide. Faint, burning taste.
Indophenol test: heat with a few cc. of cone. HC1 and evap-
orate to about 20 drops. Cool, add aqueous phenol solution
and then calcium hypochlorite solution drop by drop. Mix-
ture, shaken with excess of ammonia, becomes dirty red to
blue-violet and blue.
Phenylisocyanide test: boil with KOH + Aq and then add
a little chloroform. Odor of phenylisocyanide.
Isolation of aniline: boil several minutes with alcoholic KOH,
dilute with water and extract with ether. Evaporation of
solvent leaves oily drops of aniline. Dissolve in water and test
with calcium hypochlorite.
Phenacetine. Tasteless. Gives indophenol but not phenyl-
isocyanide test.
Cone. HNO 3 : yellow color when cold. Dil. HNO 3 dissolves
with yellow or orange-yellow color, if heated. Yellow nitro-
phenacetine crystallizes as saturated solution cools.
142 DETECTION OP POISONS
Salicylic Acid. Sweet, acidulous, harsh taste.
FeCl 3 + Aq: aqueous solutions colored blue-violet; if dilute,
more of a red- violet.
Millon's test: red color upon warming.
Br 2 _|_ Aq: yellowish white, crystalline precipitate.
Veronal. Bitter. Crystalline.
Dissolve ether residue in very little NaOH + Aq or (H 4 N)-
OH + Aq, filter and acidify filtrate with dil. HC1. Veronal
crystallizes. Wash with a little cold water, dry and determine
melting-point (187-188). The crystals mixed with pure
veronal should have same melting-point.
Antipyrine. Mild, bitter taste. Examine aqueous solution
of ether residue for antipyrine.
FeCl 3 + Aq: red color.
HNO 3 : green color with 1-2 drops of fuming acid. Heat
and a few more drops of fuming acid change green color to red.
Most of the antipyrine in ether extract of alkaline solution
(see B).
Caffeine. Faintly bitter.
Cl2 + Aq: evaporated upon water-bath with saturated
Cl2 + Aq, gives red-brown residue which turns purplish red
moistened with very little (H 4 N)OH -f Aq.
Most of the caffeine in ether extract of alkaline solution
(seeB).
Cantharidin. 1 Rhombic leaflets from ether solution.
Physiological test: triturate residue with few drops of almond
oil and test mixture as vesicant by applying to upper part of
the arm.
B. Ether Extract of Alkaline Solution may Contain :
Coniine. Yellow oil drops with penetrating odor.
Cold saturated aqueous solution becomes milky when
warmed.
1 Cantharidin is taken up in Chapter IV of this book upon page 203. Ether
extracts this compound from acid solution but it dissolves with difficulty in this
solvent (o.n : 100 at 18).
NON-VOLATILE POISONS 143
Spontaneous evaporation with a drop of HC1 gives coniine
hydrochloride as doubly refractive crystals which are needle
or prism-shaped and sometimes in star-like clusters.
Physiological test: paralysis of peripheral nerves.
Nicotine. Liquid. Remains dissolved in residual water
upon evaporation of ether and has fajnt tobacco odor.
Melzer's test: red color, heated with 2-3 cc. of epichlorohy-
drin.
Schindelmeiser's test: nicotine, after standing several hours
with a drop of formaldehyde solution, gives an intense red color
with a drop of cone. HNOs.
Roussin's test: ether solution of iodine after some time pro-
duces ruby-red, crystalline needles.
Aniline. Yellow, reddish or brownish oil drops from evapora-
tion of ether extract. (See page 60, "Synopsis of Group I,"
for further details.)
Veratrine. Cone. H 2 SO 4 : soluble with yellow color, gradually
changing to orange, then to red and finally to cherry-red. Gen-
tle heat hastens these changes. Solution at first shows greenish
yellow fluorescence.
Froehde: same color changes as with cone. H 2 SO 4 .
Cone. HChvery stable red color when heated in test-tube
upon water-bath.
Weppen's test: mixed with 6 times the quantity of cane-
sugar + a few drops of cone. H 2 SO 4 , gradually becomes green
and finally blue. Cone. H 2 SO 4 containing furfural may be
used instead.
Vitali's test: same as for atropine (see below).
Strychnine. Fine, crystalline needles having a very bitter
taste upon evaporation of ether extract.
Oxidation test: colorless solution in cone. H 2 SO 4 becomes
evanescent blue or blue-violet with a little solid K 2 Cr 2 O7.
Same color given by Mandelin's reagent but more permanent.
Brucine. Cone. HNO 3 : dissolves with blood-red color soon
changing to reddish yellow and yellow. Dilution of yellow
solution in a test-tube with a little water and addition drop by
drop of dilute SnCl 2 + Aq changes yellow to violet.
144 DETECTION OF POISONS
Careful addition of solution in dil. HNO 3 to cone. H 2 SO 4 as
upper layer produces red or yellowish red zone.
Atropine. Vitali's test: evaporated upon water-bath in por-
celain dish with a little fuming HNO 3 , gives yellowish residue
which becomes violet when moistened with alcoholic KOH.
Hyoscyamine and scopolamine also give this test. Strychnine
and veratrine behave similarly.
Para-Dime thylamino-Benzaldehyde test: intense red- violet
color. Hyoscyamine and scopolamine also give this test.
Physiological test: enlargement of pupil of eye caused by a
single drop of solution i : 130,000.
Cocaine. Free base, precipitated by KOH -f Aq from not
too dilute cocaine salt solution, forms oil drops soon becoming
solid and crystalline.
Benzoyl group: heat 5 minutes in a test-tube upon boiling
water-bath with i cc. cone. H 2 SO 4 . Odor of methyl benzoate
upon addition of 2 cc. of water. Upon cooling, benzoic acid
separates. This acid, washed and dried, recognized by melting-
point (120) and by tendency to sublime.
Physiological test: anesthesia of the tongue.
Codeine. Cone. H 2 SO 4 : soluble without color. Reddish or
more bluish upon long standing, or at once upon gentle warming.
Oxidation: deep blue or blue- violet, when warmed with
cone. H 2 SO 4 and KH 2 As0 4 , or with a little FeCl 3 + Aq.
Froehde: yellowish color soon changing to green and to blue
upon gentle warming.
Sugar test: purple-red color upon gently warming with cone.
H 2 SO 4 and a little cane-sugar. Due to fuifural formed.
Formalin test: dissolves in cone. H 2 SO 4 containing formalde-
hyde with reddish violet color soon changing to permanent blue-
violet.
Pellagri's test: given by codeine (see apomorphine, page 146).
Hydrastine. Froehde: dissolves with fairly permanent blue-
color later changing to brown.
Mandelin: dissolves with reddish color gradually changing
to orange-red.
Fluorescence: intense blue fluorescence (characteristic) upon
NON-VOLATILE POISONS 145
shaking dil. H2SO4 solution with very dilute KMnO 4 + Aq
added carefully drop by drop.
Quinine.- Amorphous varnish having very bitter taste from
ether.
Fluorescence: blue fluorescence in dil. H 2 SO 4 .
Thalleioquin test: emerald-green color, upon adding i cc.
saturated C1 2 + Aq to dilute acetic acid solution and then at
once excess of (H 4 N)OH + Aq drop by drop.
Herapathite test: heat to boiling with 10 drops of mixture
(30 drops acetic acid + 20 drops absolute alcohol + i drop dil.
H 2 SO 4 ) and add i drop alcoholic iodine solution (1:10).
Shining, olive-green leaflets, appearing cantharides-green by
reflected light.
Antipyrine. Freely soluble in water. Neutral. Mildly
bitter.
Dissolve ether residue in little water and test for antipyrine as
directed in A (see page 142).
Pyramidone. Fine needles from ether. Freely soluble in
water. Neutral.
FeCls + Aq: aqueous solution blue- violet or more red- violet.
HNOs: fuming acid renders aqueous solution blue to blue-
violet.
Caffeine. Concentric clusters of shining needles from ether.
Mild, bitter taste. Fairly soluble in water. Neutral.
Apply tests described under A (see page 142).
Physostigmine. (H 4 N)OH + Aq: evaporated with (H 4 N)-
OH + Aq, gives blue residue soluble in alcohol with same color.
Physiological test: causes contraction of pupil of eye.
Narcotine. Not bitter. Neutral.
Froehde: soluble with green color. A concentrated reagent
(0.05 gram (H 4 N) 2 MoO 4 to i cc. cone. H 2 SO 4 ) gives greenish
color at first which gradually changes to cherry-red and to a
blue from margin toward center.
Erdmann: soluble with fine red color.
Papaverine. Tasteless, colorless, neutral prisms.
Cone. H 2 S0 4 : pure alkaloid soluble without color. Heat
produces dark violet color.
146 DETECTION OF POISONS
Froehde: soluble with green color, soon changing when
warmed to blue, violet and finally cherry-red.
HNO 3 -H 2 SO 4 test: cone. H 2 SO 4 containing HNO 3 , or
cone. HNO 3 itself, gives a dark red solution.
Thebaine. Tasteless, colorless, a'lkaline prisms.
Cone. H 2 SO 4 : soluble with deep red color. Froehde and
Erdmann behave similarly.
C. Ether Extract 1 of Ammonia Solution may Contain:
Apomorphine. Residue amorphous and usually green.
H^SCVHNOs test: solution in cone. H 2 SO 4 colored evanes-
cent violet, then reddish yellow or orange by drop of cone.
HNO 3 .
Froehde: soluble with green or violet color.
Pellagri's test: dissolve in dil. HC1, add excess of NaHCO 3 ,
shake well and add i drops alcoholic iodine solution. Blue or
emerald-green color soluble in ether with violet color.
Wangerin's test: 1-2 drops K 2 Cr 2 O 7 + Aq (0.3 per cent.),
added to apomorphine hydrochloride solution, gradually pro-
duces dark green color. Chloroform added becomes violet.
Addition of dil. SnCl 2 + Aq produces pure indigo-blue color.
D. Chloroform Extract of Ammonia Solution may
Contain :
Morphine. Very bitter. Usually amorphous. Rarely crys-
talline.
Froehde: soluble with violet color gradually changing to
dirty green and finally to pale red.
Formaldehyde-H 2 SO 4 : soluble with purple-red color later be-
coming blue-violet and almost pure blue.
Husemann's test: dissolve in cone. H 2 SO 4 , heat over very
small flame until abundant white fumes appear, cool and add i
drop cone. HNO 3 . Very evanescent, red-violet color which
soon changes to blood-red or reddish yellow.
1 Unless the tartaric acid and alkaline solutions, as well as their ether extracts,
behave as described on page 126, that is to say, have a green or red color, omit
this extraction.
NON-VOLATILE POISONS 147
Pellagri's test: see apomorphine.
FeCl 3 +Aq: dissolve in few drops very dilute HC1, evaporate
to dryness upon water-bath, dissolve in little water and add
drop FeCl 3 +Aq. Blue color.
Bismuth test: dissolve in cone. H 2 S0 4 and sprinkle bismuth
subnitrate on surface of solution. Dark brown color.
Antipyrine and Caffeine. Being soluble in ether with some
difficulty, but readily soluble in chloroform, these substances
may appear in the residue from D, if they have not been pre-
viously completely extracted with ether.
Narceine. I 2 test: blue color with I 2 +Aq.
Resorcinol-H 2 SO 4 test: dissolves in resorcinol-H 2 SO 4 , giving
intense yellow solution which becomes carmine-red or cherry-
red, if warmed upon the water-bath and stirred.
Tannin-H 2 SO 4 test: dissolves in tannin-H 2 SO 4 , giving yellow-
ish brown solution which becomes pure green, if warmed upon
the water-bath.
CHAPTER III
METALLIC POISONS
Destruction of Organic Matter
The analyst cannot rely upon tests for poisonous metals, if
animal or vegetable matter is present. Consequently complete
destruction of interfering organic substances is absolutely
essential to success. Description of a few of the more important
methods used for this purpose will suffice.
i. Fresenius-v. Babo Method 1
The residue left after removal of volatile poisons by steam
distillation may be used in this part of the analysis, as it must
contain poisonous metals if any are present.
A portion of the original material, 2 previously finely chopped
and well mixed in a large flask with enough water to produce a
fluid mass, may also be used. According to the quantity of
material, add 10, 20 or 30 cc. of pure concentrated hydrochloric
acid. 3 Finally add 1-2 grams of potassium chlorate, shake well
and set the flask upon a boiling water-bath. Nascent chlorine
should come into contact with the material as intimately as
possible.
When the mixture is hot enough, add 0.3-0.5 gram of potas-
sium chlorate at 5 minute intervals and shake the flask fre-
quently. Continue in this manner, until most of the organic
matter is dissolved and the solution is pale yellow. Further
1 Annalen der Chemie und Pharmazie 49, 306 (1844).
1 Cadaveric material should be divided as finely as possible, then brought to a
thin mixture by stirring with 12.5 per cent., arsenic-free hydrochloric acid and
heated with frequent shaking with 1-2 grams of potassium chlorate as directed
above. If the material is heated on the water-bath in a porcelain dish, it should
be stirred constantly.
3 In laboratory experiments 5-10 cc. cone, hydrochloric acid is usually suffi-
cient. A large excess of hydrochloric acid should be avoided.
148
METALLIC POISONS
149
addition of potassium chlorate and longer heating should
produce no real change. Fat especially resists the action of
chlorine.
When organic matter is completely destroyed, dilute with
hot water, adding a few drops of dilute sulphuric acid to pre-
cipitate possible barium,
shake and pour the liquid
through a wetted filter. If
the excess of free hydro-
chloric acid is not too large,
satuiate the filtrate direct
with hydrogen sulphide as
directed on page 152. Other-
wise, evaporate the solution
in" a porcelain dish upon the
water-bath nearly to dryness
to remove most of the free
hydrochloric acid. This step
frequently gives rise to a dark
brown color which a few
crystals of potassium chlorate
will discharge. In testing for
lead, cadmium and copper, it
is advisable to evaporate,
because hydrogen sulphide
precipitates the first two
metals incompletely, or not
at all, from solutions contain-
ing too much hydrochloric p IG . I2 .
acid.
An alternative procedure consists in removing part of the
free hydrochloric acid from the filtrate, obtained after treat-
ment with hydrochloric acid and potassium chlorate, by first
evaporating to smaller volume and then adding ammonium
hydroxide solution until alkaline. Add dilute nitric acid until
the solution is faintly acid and saturate with hydrogen sulphide
(seepage 152).
150 DETECTION OF POISONS
The residue upon the filter may contain silver chloride,
barium sulphate and lead sulphate in addition to fat. Examine
as directed under "Metallic Poisons IV" (see page 170).
H. Thorns 1 destroys organic matter in the apparatus shown
in Fig. 12. Oxidation is carried on in an ordinary fractioning
flask (A) with the tubulus (B) bent upward. A separating
funnel (C), held in the neck of the flask by a stopper, contains
an aqueous solution of potassium chlorate (i : 20) saturated at
room temperature. The organic matter is in the flask as a
thin mixture with 12.5 per cent, hydrochloric acid. Add about
i gram of solid potassium chlorate and warm the flask on a
boiling water-bath. When the mass in the flask is warm, let
the potassium chlorate solution run in drop by drop and shake
-constantly. Care must be taken not to add too much of this
solution at once; otherwise the procedure is identical with that
previously described.
Notes. Potassium chlorate and hydrochloric acid evolve chlorine (a and @),
part of which acts upon the organic material and part in contact with water forms
oxygen and oxygen-acids of chlorine (HOC1) (<y and S) which are strong oxidizing
agents:
(a) KC10 3 + HC1 = HC10 3 + KC1,
03) HC10 3 + S HC1 = 3 C1 2 + 3 H 2 0,
(7) C1 2 -I- H 2 = 2 HC1 + O,
(5) C1 2 + H 2 0<=iHOCl + HC1.
White Arsenic (As 2 O 3 ) in a mixture probably cannot be volatilized as arsenic
trichloride (AsCl 3 ) in the procedure described but is oxidized to non- volatile
arsenic acid (H 3 AsO 4 ) :
As 2 O 3 + aH 2 O + 2 C1 2 = As 2 6 + 4HC1,
As 2 O 8 + 3H 2 O = 2 H 3 AsO 4 .
There always remains, even after the most thorough treatment with hydro-
chloric acid and potassium chlorate, an insoluble white residue wholly unaffected
by the action of chlorine. This is the case, especially after the oxidation of
vegetable substances or cadaveric material. This treatment converts a portion
of the organic matter into volatile compounds (chloranil?) which have a sharp
odor and attack the mucous membranes. For this reason destruction of organic
matter should take place in a hood with a good draft.
Treatment as described with hydrochloric acid and potassium chlorate converts
metallic poisons into inorganic salts, usually chlorides and sulphates. These
either remain in solution or appear as precipitates (AgCl and BaSO 4 ). Protein
substances, present in all animal and vegetable organisms, precipitate many
y metals, as mercury, silver, lead, copper and zinc, from solutions of their
1 H. Thorns, "Einfuhrung in die praktische Nahrungsmittel Chemie," Leipzig,
1899. Published by S. Hirzel, Leipzig, 1899. Figure 64, page 153.
METALLIC POISONS 151
salts. These metals are then in the form of metallic albuminates, some of which
dissolve in water with great difficulty and are very stable. Usually these metal-
lic protein compounds must receive further treatment before it is possible to
detect the metal. Many organic acids, as tartaric acid, and carbohydrates inter-
fere more or less with the detection of heavy metals. In combination with these
organic substances heavy metals are like copper in potassium cuprocyanide
(K 4 Cu2(CN) 6 ), which neither sodium hydroxide nor hydrogen sulphide will pre-
cipitate because it is electrolytically dissociated in solution in part as follows:
K 4 Cu 2 (CN) 6 ^4K- + Cu 2 (CN) 6 "".
In other words, the solution does not contain cuprous ions. If potassium cupro-
cyanide is heated with hydrochloric acid and potassium chlorate, copper passes
into solution as cupric chloride. The reagents mentioned above now precipitate
copper, for cupric chloride ionizes as follows:
CuCl 2 <=*Cu" + 2d'.
And the solution how contains cupric ions.
The detection, therefore, of these metallic poisons by the usual ionic reactions
requires a procedure which permits the analyst to bring about complete destruc-
tion of interfering organic substances. The metals in question are thus con-
verted into inorganic salts.
Potassium chlorate acts best only in strong hydrochloric acid solution. Conse-
quently this acid should always be in excess. If the mass becomes too thick at
any time during heating, it should be diluted with water or dilute hydrochloric
acid. Also the contents of the flask should be well shaken during treatment with
potassium chlorate, to prevent a large quantity of this salt from collecting upon
the bottom of the flask. Such an occurrence may cause an explosion due to
formation of the exceedingly unstable dioxide of chlorine (CIO*). 1
The author employs in such analyses 12.5 per cent, hydrochloric acid (sp. gr.
1.061), saturated with hydrogen sulphide and kept in a loosely stoppered bottle.
This insures precipitation of the final traces of arsenie sometimes present even in
the purest commercial acid. 2 Before being used, this acid is filtered through ash-
free paper to remove precipitated sulphur which may contain arsenic sulphide.
Cadaveric material, heated with hydrochloric acid and potassium chlorate, is
dissolved rather easily. An experiment, in which 100 grams of stomach and
duodenum, 20 grams of stomach contents, 75 grams of kidney and 200 grams of
liver (in all 395 grams) were treated as described, required about i hour for com-
plete solution. The insoluble part was collected upon a filter and washed. It
was amorphous, gummy, yellowish white and greasy. After being dried upon
a porous earthen plate, it weighed 52 grams. Dried at 100, it weighed only
32 grams.
2. Sonnenschein-Jeserich Method
This method requires the use of pure free chloric acid instead
of potassium chlorate. Place the finely divided material in a
1 (a) KC10 3 + HC1 = HC10 3 + KC1,
(0)3
) 3 HC10 3 = HC10 4 + 2C10 2 + H 2 O.
2 The use of electrolytic hydrochloric acid (see page 241) avoids the necessity
of this purification. Tr.
152 DETECTION OF POISONS
large flask and dilute with water. Add a few cc. of chloric acid
and warm slowly and cautiously upon the water-bath. As soon
as the mass swells and becomes porous, gradually add small
portions of hydrochloric acid. Even a considerable quantity
of cadaveric material will dissolve in 2-3 hours. Water lost
by evaporation should be replaced occasionally, otherwise the
reaction may take place with explosive violence. In other le-
spects, the product of the reaction should be treated as already
described.
3. C. Mai's Method 1
Mix the finely divided material with dilute hydrochloric acid
(i : 12) until thin. Add a little potassium chlorate and heat
over a free flame, adding from time to time small quantities of
potassium chlorate (0.2 gram). Cool as soon as liquefaction of
the mass is complete. Fat separates and usually can be re-
moved easily from the liquid. Heat this fat once or twice with
very dilute nitric acid, filter and add the filtrate to the main part
of the liquid. Continue heating the latter, adding small quan-
tities of ammonium persulphate, (H 4 N) 2 S 2 O8, until the liquid is
clear and light yellow. Filter and saturate the filtrate as usual
with hydrogen sulphide. Ammonium persulphate is a powerful
oxidizing agent and also adds nothing non-volatile to the
liquid.
Examination of Filtrate for Metallic Poisons
Precipitation by Hydrogen Sulphide
A solution properly prepared according to the Fresenius-v.
Babo or any other method, freed from excess of hydrochloric
acid and filtered, should have only a faint yellow color. 2 Heat
such a solution in a flask upon the water-bath and saturate
with arsenic-free hydrogen sulphide. 3 Pass hydrogen sulphide
x Zeitschrift fur Untersuchung der Nahrungs- und Genussmittel 5, 1106
(1902).
2 Chromium in not too small quantity imparts more or less of a green color
both to the solution and the nitrate from the hydrogen sulphide precipitate,
owing to the presence of chromic chloride (CrCls).
3 Prepare arsenic-free hydrogen sulphide by saturating dilute sodium hydroxide
solution with hydrogen sulphide from crude iron sulphide and commercial hydro-
METALLIC POISONS
153
for 0.5-1 hour or longer 1 into the hot solution and continue
this treatment after the solution has been removed from the
water-bath and is cold.
Allow the solution saturated with hydrogen sulphide to stand
in the loosely stoppered flask for several hours or until the next
day. If the solution then smells of hydrogen sulphide and
blackens a piece of lead acetate paper held over it, the next step
PIG. 13. Apparatus for Generating Arsenic-free Hydrogen Sulphide, (a)
Generator with dilute sulphuric acid; (b) Separating funnel with NaSH; (c) Wash-
bottle; (d) Solution to be saturated with H2S.
in the process may be taken. Otherwise warm the solution
once more upon the water-bath and again saturate with hydro-
gen sulphide. Finally collect the hydrogen sulphide precipi-
tate upon a small paper and wash with hydrogen sulphide water.
Examine the precipitate for arsenic, antimony, tin, mercury,
lead, copper, bismuth and cadmium (Metallic Poisons I and II)
chloric acid. Pour this sodium hydro-sulphide (NaSH) solution into a separating
funnel and add slowly to dilute sulphuric acid (1:4). The generation of the gas
can be carried on in the apparatus shown in Fig. 13.
1 In laboratory experiments treatment with hydrogen sulphide may be short-
ened somewhat. A Kipp generator in which the gas is prepared from iron
sulphide and hydrochloric acid may be used.
154 DETECTION OF POISONS
and the filtrate from this precipitate for chromium and zinc
(Metallic Poisons III).
Vegetable and animal substances, after treatment with
hydrochloric acid and potassium chlorate, frequently give
liquids yielding colored precipitates 1 with hydrogen sulphide
even in the absence of the metals mentioned above. Such
precipitates consist largely of organic sulphur compounds.
Consequently, if hydrogen sulphide 4 produces such a colored
precipitate in acid solution, it is not final proof of the presence
of a metallic poison. Also without further examination it is
impossible to decide from the color of the hydrogen sulphide
precipitate as to the presence of a particular metal.
Complete Precipitation. Before testing for chromium and
zinc in the filtrate from the hydrogen sulphide precipitate, add
about 10 times the volume of strong hydrogen sulphide water
to a small portion of the solution, stir well and let stand several
minutes. Unless a colored precipitate appears, the metals in
question (Metallic Poisons I and II) have been completely
removed and the filtrate may then be further tested for chrom-
ium and zinc (Metallic Poisons III). Otherwise, first dilute
the entire filtrate from the hydrogen sulphide precipitate with
water and again saturate with hydrogen sulphide. In presence
of much hydrochloric acid, lead and cadmium are incompletely
precipitated by hydrogen sulphide (see above).
Treatment of Hydrogen Sulphide Precipitate with Ammonia
and Yellow Ammonium Sulphide. Extract the thoroughly
washed hydrogen sulphide precipitate, while still moist, upon
the filter with a hot mixture of approximately equal parts of
ammonia and yellow ammonium sulphide. Heat about 5-10
cc. of the mixture of ammonia and yellow ammonium sulphide
to boiling and drop the solution over the precipitate upon the
filter. Reheat the filtrate and again pour over the precipitate.
Repeat this operation several times. Finally wash the filter
1 Repeated treatment with potassium chlorate and hydrochloric acid dissolves
thoroughly washed casein and fibrin almost completely and gives a nitrate from
which hydrogen sulphide precipitates dirty yellow to brownish substances.
These products are amorphous and contain organic sulphur compounds together
with much free sulphur.
METALLIC POISONS 155
with a few cc. of a fresh mixture of ammonia and yellow ammo-
nium sulphide. Test the entire filtrate for arsenic, antimony,
tin and copper 1 = Metallic Poisons I. Test the residue upon
the filter for mercury, lead, copper, bismuth and cadmium =
Metallic Poisons II.
METALLIC POISONS I
Examination of the Part of the Hydrogen Sulphide Precipitate Soluble
in Arnmonia-Ainrnoniurn Sulphide
Arsenic, Antimony, Tin, Copper
Use the solution prepared as described by treating the hydro-
gen sulphide precipitate with a hot mixture of ammonia and
yellow ammonium sulphide. This solution is usually dark
brown owing to dissolved organic substances. 2 Evaporate the
solution to dryness in a porcelain dish upon the water-bath.
Moisten the cold residue with fuming nitric acid and again
evaporate. Then intimately mix the residue with about 3 times
its volume 3 of a mixture of i parts of sodium nitrate and i part
of dry sodium carbonate. Thoroughly dry this mixture upon
the water-bath and introduce small portions at a time into a
porcelain crucible containing a little fused sodium nitrate
heated to redness. After the final addition, heat the crucible
1 Copper sulphide (CuSj is somewhat soluble in hot yellow ammonium sul-
phide. An ammonium sulphide solution containing copper, treated as described
on page 156, yields copper oxide which gives the melt a more or less gray or black
appearance. If the melt is extracted with water, the residue contains black
copper oxide with stannous oxide and sodium pyro-antimonate. To detect cop-
per, dissolve the black residue in a little hot dilute hydrochloric acid and divide
the solution into two parts. Add ammonia to i part until alkaline. The solu-
tion is blue, if copper is present. Add potassium ferrocyanide solution to the
other part. A brownish red precipitate of cupric ferrocyanide (Cu2Fe(CN)g)
appears, if copper is present.
2 In absence of metals the appearance of dark colored precipitates (see above),
when the hydrochloric acid solution is treated with hydrogen sulphide, should not
be misunderstood. Such precipitates are due to organic substances soluble with
a dark brown color in a hot mixture of ammonia and yellow ammonium sulphide.
3 In most laboratory experiments 3 grams of a mixture of 2 grams of sodium
nitrate and i gram of sodium carbonate are sufficient. A large excess of sodium
nitrate should be avoided.
156 DETECTION OF POISONS
a short time, introducing possibly a little more sodium nitrate,
until the fused mass is colorless. In presence of copper the
melt is gray or grayish black from copper oxide. Sodium
arsenate, sodium pyro-antimonate, sodium stannate, as well as
stannic oxide and copper oxide, may also be present. Soften
the cold melt with hot water and wash into a flask. Add a
little acid sodium carbonate to the clear or cloudy liquid to de-
compose the small quantity of sodium stannate possibly in solu-
tion and precipitate all the tin as stannic oxide and then filter.
The filtrate (A) contains any arsenic present as sodium arsen-
ate (Na2HAs04) and the residue upon the filter (B) may con-
tain 1 sodium pyro-antimonate (Na2H2Sb2Oy), stannic and copper
oxides.
Examination of Filtrate A for Arsenic
Arsenic is isolated as the element. Positive proof of the pres-
ence of the poison is thus afforded. Two methods are in use for
this purpose, namely, the Marsh-Berzelius and the Fresenius-v.
Babo method. Both are very accurate and exclude any con-
fusion of arsenic and antimony.
Chapter V gives the details for detecting arsenic by the very
delicate biological test, which requires the use of certain moulds,
and also for the electrolytic separation of arsenic at the cathode
as arsine.
I. Marsh-Berzelius Method
Principle. Nascent hydrogen converts oxygen compounds
of arsenic, arsenious and arsenic acids, as well as arsenites and
arsenates into arsine, AsH 3 :
As 2 3 + i 2 H = 2 AsH 3 + 3 H 2 0,
As 2 8 .3H 2 2 + i6H = 2 AsH 3 + 8H 2 O.
At a red heat arsine is decomposed into metallic arsenic and
hydrogen:
AsH 3 = As + 3 H.
This reaction represents the formation of the arsenic mirror.
1 Even in the absence of the substances mentioned under B, a small insoluble
residue usually appears. This may come from the porcelain crucible, the glazing
of which is slightly attacked in the fusion with sodium nitrate and carbonate.
2 As 2 O 6 .3H 2 O is the dualistic method of writing 2 H 3 AsO 4 .
METALLIC POISONS 157
Also hydrogen containing arsine burns with a bluish white
flame (a). Depress a piece of cold porcelain upon such a flame.
Hydrogen will burn but a deposition of metallic arsenic takes
place (|8). This is the so-called arsenic spot:
(a) 2 AsH 3 + 30 2 = As 2 3
08) 2 AsH 3 + 30 = 2As + 3 H 2 0.
Hydrogen containing arsine precipitates black metallic silver.
if passed into dilute silver nitrate solution. The solution con-
tains arsenious acid:
AsH 3 + 3 H 2 + 6AgN0 3 = H 3 AsO 3 + 6HNO 3 + 6Ag.
Procedure. First acidify " Filtrate A," prepared as described
(page 156) and possibly containing sodium arsenate, with dilute
arsenic-free sulphuric acid. Evaporate this solution in a porce-
lain dish upon an asbestos plate over a small free flame. Add a
few drops of concentrated sulphuric acid, to expel completely
any nitric acid possibly present in the residue, and heat until
copious white fumes of sulphuric acid appear. The residue 1
in the porcelain dish is a thick colorless liquid having a strong
acid reaction. Arsenic, if present, is in the form of arsenic acid
which when cold frequently solidifies to a white crystalline
mass. Examine the solution of this residue in the Marsh ap-
paratus for arsenic. The same solution may also be used in
testing for arsenic electrolytically (see pages 233 and 237).
Marsh Apparatus. Place 30-40 grams of pure arsenic-free
zinc 2 (granulated or in small rods) in the reduction flask A of
the Marsh apparatus (Fig. 14). Pour cold dilute arsenic-free
sulphuric acid upon the metal. This acid should 'contain
15-16 per cent, of H 2 S0 4 . 3 Control the temperature of the
solution, which should not rise much during the analysis, by
1 To insure complete removal of nitric acid, test a few drops of this residue with
ferrous sulphate and sulphuric acid.
2 The passage of hydrogen from 15-20 grams of zinc, treated with dilute
arsenic-free sulphuric acid, through the strongly heated ignition-tube C of the
Marsh apparatus should not give a trace of arsenic after i hour. The metal is
then pure enough for use. Bertha spelter from the New Jersey Zinc Company
will meet such a test.
3 Add i volume of pure arsenic-free concentrated sulphuric acid to 5 volumes of
distilled water. This diluted acid when cold is suitable for use in the Marsh test.
158
DETECTION OF POISONS
generating hydrogen slowly. Otherwise, there is danger of
partial reduction of sulphuric acid to sulphur dioxide and then
to hydrogen sulphide which interferes more or less with the
detection of arsenic. Place the reduction flask A in a dish of
cold water, if the acid becomes too warm.
Certain precautions are necessary in using the Marsh ap-
paratus.
1. Have the apparatus absolutely tight.
2. Expel air completely before igniting hydrogen. To tell
when this point is reached, collect hydrogen in a dry test-tube
FIG. 14. Marsh Apparatus, (a) Hydrogen-generator; (&) Chloride of cal-
cium drying-tube; (c) Hard glass tube; (d) Arsenic mirror.
until it ignites without detonation when carried to a flame. If
the hydrogen stands this test, ignite the gas at the end of igni-
tion-tube C. There is no danger of an explosion within the
apparatus. If the apparatus is tight and the evolution of
hydrogen is not too rapid, it requires about 8 minutes to expel
the air.
3. Test the hydrogen to insure its entire freedom from arsenic.
Neither the arsenic mirror nor spot appears.
If the hydrogen is arsenic-free, gradually introduce the
perfectly cold sulphuric acid solution, containing arsenic as
arsenic acid (page 157), in small portions into reduction-flask A.
At the same time heat ignition-tube C to redness just back of
METALLIC POISONS 159
the capillary tube. If the solution contains arsenic, the gas
generated consists of a mixture of arsine (AsH 3 ) and hydrogen.
A shining mirror of metallic arsenic appears, often in a few
minutes, just beyond the point of ignition. Traces of arsenic
require considerable time before a brown or brownish black
film appears. A piece of white paper held behind the tube
brings out clearly even a minute arsenic mirror.
Remove the flame from ignition- tube C. If arsenic is present,
the hydrogen flame becomes bluish white. At the same time
white fumes of arsenious oxide (As 2 O 3 ) arise from the flame.
To produce the lustrous, brownish black spot (arsenic spot),
depress a cold porcelain dish upon the hydrogen flame.
Arsine has an exceedingly characteristic, garlic-like odor.
Extinguish the hydrogen flame and allow, the gas to escape. 1
The odor is evident even when the hydrogen contains traces
of arsine.
A third method of detecting arsenic by the Marsh apparatus
consists in extinguishing the hydrogen flame and passing the
gas into dilute silver nitrate solution. Arsine darkens this
solution, producing a black precipitate of metallic silver. The
solution contains arsenious acid and free nitric acid. (See
reaction, page 157.)
Filter through a double paper to remove silver and carefully
neutralize the filtrate with a few drops of very dilute ammonium
hydroxide solution. If the solution is neutral, it is possible
to obtain a yellowish white precipitate of silver arsenite
(Ag 3 AsO 3 ) but this compound dissolves easily in ammonium
hydroxide solution and in nitric acid.
Extinguish the flame at the end of .reduction-tube C and hold
over the tube a strip of paper moistened with concentrated
silver nitrate solution (i : i). A yellow stain appears, if the
hydrogen contains arsine. A drop of water added to this
yellow spot changes the color to black. This is Gutzeit's
arsenic test (see page 163).
1 The detection of arsine by other tests is so easy that it seems somewhat
superfluous to confirm its presence in this way in view of its very poisonous
properties. Tr.
160 DETECTION OF POISONS
Differences Between Arsenic and Antimoriy Spots and Mirrors
Nascent hydrogen reduces various antimony compounds (SbCl 3 , Sb 2 Os,
HSbO 3 , KSbOC 4 H 4 O 6 , etc.) producing the colorless gas stibine (SbH 3 ). The
behavior of this compound in the Marsh apparatus closely resembles that of
arsine, for it gives a spot and mirror, and precipitates black silver antimonide
(AgsSb) but not metallic silver, if passed into silver nitrate solution.
The procedure employed in preparing material for the Marsh test (see page
155) separates arsenic from antimony and excludes the possibility of the two met-
als appearing in the Marsh test at the same time. Since the identification of
arsenic minors and spots by other tests is important, the differences between
arsenic and antimony should be pointed out. The suspicion that antimony is
present often necessitates other confirmatory tests (see Antimony, page 164).
Introduce the solution into the Marsh apparatus and produce the antimony
spot and mirror.
The differences between arsenic and antimony spots and mirrors are:
1. The arsenic mirror has a high metallic luster. It is brownish black and
volatile. Owing to this latter property, it sublimes easily when heated in a
stream of hydrogen. In the case of the antimony mirror, which appears on both
sides of the flame, the metal in contact with the heated glass fuses and is silver
white. But in those places removed from the flame it is almost black and has
hardly any luster. Stibine decomposes at a temperature much below that
required for arsine. This fact explains the deposition of this metal on both sides
of the flame. Antimony volatilizes at a high temperature and consequently
sublimes with difficulty.
2. The arsenic spot, if not too heavy, is brownish black or brown and lustrous.
It dissolves readily in sodium hypochlorite solution, forming arsenious acid:
3H 2 + 2 As + 3 NaOCl = 2H 3 AsO 3 + sNaCl.
The antimony spot is dull, velvet-black and without luster. A thin film of
antimony is never brown but has a dark, graphite-like appearance. It is insolu-
ble in sodium hypochlorite solution. Only a freshly prepared solution, however,
should be used in this test, since Vaubel and Knocke 1 have shown that an old
solution will dissolve the antimony spot owing to presence of sodium chlorite,
formed as shown in the reaction from sodium hypochlorite :
2NaOCl = NaClO 2 + NaCl
3. A drop of concentrated nitric acid, or moist chlorine, at once dissolves the
arsenic spot forming arsenic acid. Neutralize with ammonia and add silver
nitrate solution. A reddish precipitate of silver arsenate (Ag 3 AsO 4 ) appears.
Nitric acid, or moist chlorine, also dissolves the antimony spot but silver
nitrate does not produce a colored precipitate.
4. Gently heat the ignition-tube and pass a stream of dry hydrogen sulphide
over the arsenic mirror. Yellow arsenic trisulphide (As 2 S 3 ) appears. The
antimony mirror becomes brownish red to black (Sb 2 S 8 ).
5. Arsine passed into silver nitrate solution precipitates black metallic silver
and the filtrate from such a precipitate contains arsenious acid. But stibine
precipitates black silver antimonide (Ag 3 Sb) and the filtrate does not contain a
1 Chemical Abstracts 10, 1147 (1916).
METALLIC POISONS
161
trace of antimony since the precipitation of black Ag 3 Sb is complete. To detect
antimony, collect the black precipitate upon paper, wash and heat for some time
in 10-15 P er cent, tartaric acid solution. Antimony dissolves, whereas silver
remains as a grayish white residue. Add dilute hydrochloric acid to this solu-
tion and then treat with hydrogen sulphide. Antimony appears as orange-red
antimony trisulphide.
2. Fresenius-von Babo Method
Principle. Fusion of oxygen and sulphur compounds of
arsenic with a mixture of sodium carbonate and potassium
cyanide causes reduction with formation of an arsenic mirror.
As a result potassium cyanide changes to potassium cyanate
(KCNO) or potassium sulphocyanate (KSCN) :
As 2 O, + 3 KCX = As 2 + 3KCNO,
As 2 S 3 + 3KCN = As 2 + 3 KSCN.
Procedure. Use for this test the sulphuric acid solution
prepared by the method already described and containing ar-
senic in the form of arsenic acid (page 157). To reduce arsenic
PIG. 15. Fresenius-Von Babo Apparatus, (a) Carbon dioxide generator;
(b) Drying-bottle with pure, concentrated sulphuric acid; (c) Ignition-tube and
boat.
acid to arsenious acid, add a few cc. of sulphurous acid to the
solution and heat until the odor of this acid has disappeared.
Dilute this solution with water and treat with hydrogen sul-
phide. Collect the precipitate of arsenic trisulphide (As 2 S 3 )
upon a small filter and wash thoroughly. Dissolve the pre-
cipitate upon the filter in a little hot ammonium hydroxide solu-
162 DETECTION OF POISONS
tion. Evaporate this solution in a porcelain dish upon the
water-bath and heat the residue with concentrated nitric acid.
Expel the latter completely by evaporation, moisten the residue
with a little water and add enough dry sodium carbonate to
render the mixture distinctly alkaline. Dry thoroughly upon
the water-bath and triturate the residue in a mortar with several
times the quantity of a mixture of 3 parts of dry sodium car-
bonate and i part of pure potassium cyanide.
Transfer this mixture to a porcelain boat and place in an igni-
tion-tube of hard glass. Heat in a stream of carbon dioxide
(Fig. 15) dried by means of arsenic-free sulphuric acid.
To expel moisture, first heat the ignition-tube gently where
the boat is and then ignite at a bright red heat. A mirror
of arsenic appears upon the cooler part of the tube, if arsenic is
present.
B,
A.
FIG. 16. (4) Substance and fusion- mixture; (B) Arsenic mirror.
A simpler method of detecting arsenic by means of potassium
cyanide is often used. Heat the thoroughly dried material
containing arsenic (As 2 O 3 , As 2 S 3 ) in a bulb tube with a dry mix-
ture of sodium carbonate and potassium cyanide until fusion
takes place. If the tube is smaller above the bulb, the arsenic
mirror will form in the constricted area (Fig. 16).
Other Arsenic Tests
i. Bettendorff's Test. Concentrated stannous chloride solu-
tion precipitates metallic arsenic from arsenious acid cold and
from arsenic acid with heat or after long standing. This test
requires the use of a special stannous chloride solution. 1 The
solution is red to brownish red, if only traces of arsenic are pres-
ent. More than traces of arsenic produce a black precipitate of
arsenic:
As 2 3 + 6HC1 + 3 SnCl 2 = 2As + 3 H 2 O + 3 SnCl 4 ,
As 2 6 + loHCl + 5 SnCl 2 + 3 H 2 O = 2 As + 8H 2
1 See page 522 for the preparation of this reagent.
METALLIC POISONS 163
Use for this test the sulphuric acid solution obtained as de-
scribed above (page 157) which contains arsenic in the form of
arsenic acid. Bettendorff's test is not as delicate as the Marsh
test.
2. Gutzeit's Test. 1 This test permits the detection of minute
traces of arsenious and arsenic acids, as well as their salts, with
certainty. Generate hydrogen in a test-tube from arsenic-free
zinc and pure dilute hydrochloric acid. To remove any sul-
phurous acid or hydrogen sulphide, add a few drops of iodine
solution until the liquid is yellow. Then add the solution to be
tested and place a loose cotton plug in the neck of the test-tube. If
arsenic is present, the silver nitrate spot becomes lemon-yellow.
AsH 3 + 6AgN0 3 = ( 3 AgNO 3 .Ag 3 As) + 3 HNO 3 .
Gradually a brownish black border forms around the yellow
spot. A drop of water at once turns the spot black from
separation of metallic silver.
(sAgNOs.AgsAs) + 3 H 2 = 6Ag + H 3 AsO 3 + 3HNO 3 .
This is a very delicate arsenic test. One drop of o.i per cent,
potassium arsenite solution produces a distinct yellow color
upon the silver paper. Gutzeit's test is positive with even 0.05
milligram of As 2 O 3 . 2 The sulphuric acid solution containing
arsenic as arsenic acid (see page 157) may be used for this test.
But Gutzeit's test is not as characteristic of arsenic as the
Marsh test. Stibine, phosphine from phosphorus in zinc and
even hydrocarbons color the silver nitrate paper. Dry hydro-
gen sulphide also produces a yellow or yellowish green spot upon
paper moistened with concentrated silver nitrate solution.
The latter spot has a black border which gradually extends
until the entire spot becomes black. Poleck gives this yellow
compound the composition (AgNO 3 -Ag 2 S).
Detection of Antimony, Tin and Copper in Residue B
Residue B (see page 156), insoluble in water and obtained
from the fusion, may contain sodium pyro-antimonate, stannic
1 Gutzeit, Pharmazeutische Zeitung 1879, 263; and Poleck and Tiimmel,
Berichte der Deutschen chemischen Gesellschaft 16, 2435 (1883).
2 See page 240 for the application of this method to the quantitative estima-
tion of arsenic. Tr.
164 DETECTION OF POISONS
and cupric oxides. Treat this residue upon the filter with a
little hot dilute hydrochloric acid (equal parts of concentrated
acid and water). Pass this acid repeatedly through the paper
until most of the residue is dissolved. If the original color
of residue B and of the melt was gray or black, first examine a
portion of the hydrochloric acid solution for copper. Excess
of ammonia produces a blue color. Potassium ferrocyanide
solution gives a brownish red precipitate, or only a coloration
with traces of copper.
Concentrate the remainder of the hydrochloric acid solution
to a few drops in a porcelain dish upon the water-bath, and put
2 drops of this solution upon platinum foil in contact with zinc.
Antimony produces a black, tin a grayish and copper a dark
reddish brown spot upon platinum. There is little chance
of confusing the tin or copper spot with that given by
antimony.
Dilute the remainder of the hydrochloric acid solution with
water and introduce a piece of zinc. Keep the zinc in the solu-
tion, as long as hydrogen is evolved. Collect the black, metal-
lic flocks from this operation upon a small filter. Wash thor-
oughly, and gently waim with a little concentrated hydrochloric
acid. Finally, filter the solution. Antimony does not dissolve,
whereas tin passes into solution as stannous chloride (SnCl 2 ),
and is in the filtrate. Apply the tests described under tin to
this solution.
TIN
The solution, treated as described in the above analytical
procedure, contains tin as stannous chloride (SnCl 2 ). Apply
the following tests for this metal:
(a) Mercury Test. Add to a portion of the filtrate a few
drops of mercuric chloride solution. Tin precipitates white
mercurous chloride (calomel). Heat produces in addition
gray, metallic mercury, if there is a large excess of stannous
chloride.
(b) Prussian Blue Test. Add to a second portion of the
filtrate a few drops of a dilute mixture of ferric chloride and
METALLIC POISONS 165
potassium ferricyanide solutions. Tin produces a precipitate
of Prussian blue.
This test is not characteristic of tin, as many other substances capable of
reducing ferri-ferricyanide to ferri-ferrocyanide, that is to say, to Prussian blue
act in the same way.
To identify antimony further, dissolve the black flocks, in-
soluble in hydrochloric acid, in a few drops of hot aqua regia.
Expel excess of acid upon the water-bath and dilute the residue
with water. If the quantity of antimony is not too small, water
precipitates white antimony oxychloride (SbOCl). Redis-
solve this precipitate in a little dilute hydrochloric acid. Test a
portion of this solution for antimony with hydrogen sulphide.
Introduce the remainder into the Marsh apparatus and produce
the antimony spot and mirror, or test for stibine with silver
nitrate solution as described (see page 160).
METALLIC POISONS II
Detection of Metals Whose Sulphides are Insoluble in Ammonium
Sulphide
Mercury Bismuth
Lead Copper
Cadmium
That portion of the hydrogen sulphide precipitate, insoluble
in ammonium sulphide solution, may contain mercury, lead,
bismuth, copper and cadmium sulphides. Examine this pre-
cipitate according to the methods employed in qualitative
analysis. Treat a small precipitate repeatedly upon the filter
with a few cc. of warm, rather dilute nitric acid (i volume of
concentrated acid and 2 volumes of water) . Mercuric sulphide
does not dissolve, but the other sulphides pass into solution
as nitrates.
Detection of Mercury in the Residue Insoluble in Nitric Acid
Always examine that portion of the hydrogen sulphide pre-
cipitate, insoluble in nitric acid, for mercury, even when not
black! Treat this residue upon the filter with a little hot,
166 DETECTION OF POISONS
somewhat diluted hydrochloric acid, containing in solution a
few crystals of potassium chlorate and pass the acid through the
paper several times. Evaporate the filtrate to dryness in a
porcelain dish upon the water-bath, and dissolve the residue in
2-3 cc. of water containing hydrochloric acid. Filter this solu-
tion,' and examine the filtrate for mercury.
(a) Stannous Chloride Test. Add to a portion of the filtrate
a few drops of stannous chloride solution. A white precipitate
of mercurous chloride (calomel) appears, if mercury is present.
Excess of stannous chloride, especially if heat is applied, re-
duces this precipitate to gray, metallic mercury.
(b) Copper Test. Put a few drops of the filtrate upon a
small piece of bright copper. Mercury immediately deposits
a gray spot which has a silvery luster when rubbed. Wash
the copper, upon which mercury has been deposited, succes-
sively in water, ethyl alcohol and ether. Dry thoroughly and
heat in a small bulb-tube of hard glass. Mercury sublimes and
collects in small, metallic globules on the cool sides of the tube.
A trace of iodine vapor, introduced into the tube, immediately
transforms the gray sublimate into scarlet mercuric iodide
(HgI 2 ).
(c) Phosphorous Acid Test. Add to another portion of the
filtrate some phosphorous acid and warm gently. A white
precipitate of mercurous chloride (calomel) appears, if mercury
is present:
2HgCl 2 + H 2 + H 3 P0 3 = Hg 2 Cl 2 + 2HC1 + H 3 PO 4 .
(d) Precipitation of Mercuric Iodide. Add 1-2 drops of very
dilute potassium iodide solution to the remainder of the filtrate.
A red precipitate (HgI 2 ), readily soluble in excess of potassium
iodide, shows mercury:
1. HgCl 2 + aKI = HgI 2 + aKCl,
2. HgI 2 + 2 KI = K 2 HgI 4 .
Examination of the Nitric Acid Solution
The nitric acid solution may contain lead, bismuth, copper
and cadmium nitrates. Evaporate this solution in a porcelain
dish nearly to dryness and dissolve the residue in a little hot
METALLIC POISONS 167
water. If the solution contains lead, dilute sulphuric acid pro-
duces a heavy white precipitate of lead sulphate. Test the
filtrate from this precipitate for bismuth, copper and cadmium.
(a) Copper and Bismuth Tests. Excess of ammonium hy-
droxide solution, added to most of this filtrate, produces a blue
color if copper is present. A white precipitate at the same time
may be bismuthous hydroxide 1 (Bi(OH) 3 ). To detect bismuth,
wash the precipitate and dissolve upon the filter in a few drops
of hot dilute hydrochloric acid. Pour this solution into consid-
erable water. A white precipitate of bismuthous oxychloride
(BiOCl) proves the presence of bismuth. As an alternative
test, add stannous chloride to the hydrochloric acid solution and
then excess of sodium hydroxide solution. A black precipitate
of metallic bismuth appears.
(&) Potassium Ferrocyanide Test. Potassium ferrocyanide
solution precipitates copper as brownish red cupric ferrocyanide
(Cu2Fe(CN)e). Traces of copper produce a brownish red
color. There is a deposit of cupric ferrocyanide after some time.
(c) Precipitation of Metallic Copper. A bright knife blade,
or a bright iron nail, immersed for a short time in a copper solu-
tion, becomes red from a coating of metallic copper.
To detect cadmium in presence of copper, add solid potas-
sium cyanide to the blue solution, produced by ammonium
hydroxide, until the blue color is discharged. Then pass hydro-
gen sulphide through the solution. Cadmium is precipitated as
the yellow sulphide (CdS), whereas copper remains in solution
as K 4 Cu 2 (CN) 6 , potassium cuprocyanide.
When copper is absent, test for cadmium by passing hydrogen
sulphide at once into the ammoniacal solution. If a reddish
or brownish instead of a yellow precipitate appears, filter, dry
the precipitate upon the paper and heat upon charcoal in the
blow-pipe flame. Cadmium gives a brown coating.
1 Ammonium hydroxide solution does not precipitate pure bismuthous hydrox-
ide (Bi(OH)sJ from solutions of bismuth salts but a basic salt, the composition
of which depends upon the temperature and concentration of the particular
solutions.
168 DETECTION OF POISONS
METALLIC POISONS III
Detection of Chromium and Zinc
Test the filtrate from the hydrogen sulphide precipitate for
chromium and zinc. Concentrate the filtrate to about one-
third its original volume and divide this solution into two parts.
Detection of Zinc
Add enough ammonium hydroxide solution to render one-
half the concentrated filtrate alkaline. This treatment usually
gives the solution a dark color. Then add ammonium sulphide
solution in excess. This reagent almost always produces a
precipitate even when zinc is not present, since solutions, pre-
pared from animal and vegetable materials, usually contain
iron compounds and phosphates of the metals of the earths and
of the alkaline earths. When this precipitate has settled, add
acetic acid until the solution has a faint acid reaction. Stir the
mixture thoroughly, and allow it to stand for some time. The
color of the precipitate becomes lighter, because acetic acid dis-
solves sulphide of iron. Moreover, the phosphates are partly
dissolved, except ferric phosphate (FePO 4 ) which is insoluble in
acetic acid. Collect the precipitate upon a filter. ^Vash, dry
and ignite precipitate and filter in a porcelain crucible. Before
ignition, moisten the filter with concentrated ammonium ni-
trate solution. Extract the residue from ignition with 3 cc.
of boiling, dilute sulphuric acid. Filter, and divide the filtrate
into two parts.
(a) Add sodium hydroxide solution in excess to one portion
of the filtrate and shake thoroughly. Filter, to remove the
white precipitate of ferric phosphate which usually appears, and
add a few drops of ammonium or hydrogen sulphide solution to
the clear filtrate and heat. Zinc, if present, gives a white,
flocculent precipitate of zinc sulphide.
(V) Add ammonium hydroxide solution in considerable ex-
cess to the second part of the filtrate. Filter, to remove ferric
phosphate, and acidify the filtrate with acetic acid. Warm
METALLIC POISONS 169
the solution and treat with hydrogen sulphide. Zinc, if present,
appears as the white sulphide.
(c) Test further for zinc by dissolving the precipitate pro-
duced by ammonium or hydrogen sulphide (as described above
in a and b] , after it has been collected upon a filter and thor-
oughly washed, in a few drops of hot dilute hydrochloric acid.
Boil until hydrogen sulphide is expelled, and filter to remove
precipitated sulphur. Add potassium ferrocyanide solution to
the clear, cold filtrate. This precipitates Zn 2 Fe(CN) 6 , zinc
ferrocyanide, which is white, slimy and nearly insoluble in dilute
hydrochloric acid. 1
Detection of Chromium
To test for chromium 2 in the second part of the filtrate
from the hydrogen sulphide precipitate, concentrate the solu-
tion in a porcelain dish to a small volume. Then add twice the
quantity of potassium nitrate and sodium carbonate, until the
reaction is decidedly alkaline. Finally, heat this mixture until
perfectly dry. Add this dry residue in small portions to a little
potassium nitrate fused in a crucible. In fusing a large quan-
tity of material, it is advisable to use a large, bright, nickel
crucible which is especially adapted for this operation. When
fusion is complete, cool thoroughly, boil crucible and contents
with water in a porcelain dish and filter the solution. Chro-
mium colors the filtrate more or less yellow. Even mere traces 3
of chromium color the filtrate yellow. When the solution of
the melt is colorless, it is unnecessary to test for chromium. To
detect chromium when the filtrate is yellow, divide the solution
into two portions and make the following tests:
1 Excess of potassium ferrocyanide combines with zinc ferrocyanide, which is
first precipitated, and forms insoluble potassium zinc ferrocyanide:
3 Zn 2 Fe(CN) 6 + K 4 Fe(CN) 6 = 2 K 2 Zn 3 (Fe(CN) 6 ) 2 .
2 In testing for metallic poisons, chromic oxide (CraOs), which is insoluble in
acids, may be disregarded as it is not poisonous.
3 Two drops of 10 per cent, potassium chromate solution (= o.oi gram of
K 2 CrO 4 ) in 500 cc. of water produce a marked yellow color. Fifty cc. of this
solution contain o.ooi gram of K 2 CrO4 which can still be recognized by the
yellow color.
170 DETECTION OF POISONS
(a) Chrome Yellow Test. Add acetic acid in excess to
one portion of the nitrate, and boil for some time to expel
carbon dioxide and nitrous acid as completely as possible.
Then add a few drops of lead acetate solution. A yellow
precipitate of lead chromate (PbCrO 4 , " Chrome Yellow")
appears, if chromium is present. When the precipitate is
mixed with considerable lead sulphate or chloride, the color
is only yellowish. A white precipitate is due to PbSO 4 , PbCl 2
or Pb 3 (P0 4 ) 2 . When the aqueous solution of the melt is color-
less, such a precipitate is usually obtained.
Potassium nitrate, always present in the melt, lead acetate and acetic acid,
brought together in solution, produce a distinct yellow color, in which a white
precipitate may appear yellow. To eliminate this source of error, allow the
precipitate to settle, collect upon a filter and wash thoroughly. If it is pure
white, chromium is absent.
(b) Reduction Test. Add sulphurous acid to the second
portion of the yellow nitrate. The yellow color changes to
green, or greenish blue, with formation of chrome alum. This is
not as delicate as the preceding test.
METALLIC POISONS IV
Detection of Barium, Lead and Silver in the Residue from Hydrochloric
Acid and Potassium Chlorate
Wash the residue left undissolved in the treatment with
hydrochloric acid and potassium chlorate thoroughly with water
and dry in the air-closet or upon a porous plate. Then add
three times the quantity of a mixture of 2 parts of potassium
nitrate and i part of sodium carbonate and triturate in a mortar
with the filter. Gradually introduce this mixture into a hot
porcelain crucible. In this operation organic substances (fats,
fatty acids, etc.) are oxidized by potassium nitrate with con-
siderable deflagration. Finally when all the material is in the
crucible, add 0.25-0.5 gram more of potassium nitrate. Cool
the melt, soften with water, wash into a flask and pass carbon
dioxide through the turbid liquid for several minutes. This
treatment converts caustic alkali into carbonate and completely
precipitates lead which may be in solution. Then heat the
METALLIC POISONS 171
solution to boiling and let settle for some time. Collect upon
paper the sediment 1 which may contain barium carbonate, basic
lead carbonate and metallic silver. Silver gives the sediment
a gray color. Thoroughly wash the precipitate with hot water
and dissolve upon the paper in hot, rather dilute nitric acid, 2
passing the acid through the paper several times. Evaporate
this solution to dryness. Dissolve the residue in water, heat
the entire solution to boiling, and precipitate silver with dilute
hydrochloric acid. Filter, to remove silver chloride, and pass
hydrogen sulphide through the nitrate to precipitate lead.
To test for barium in the nitrate from lead sulphide, first boil
to expel hydrogen sulphide and filter to remove insoluble
matter. Then add dilute sulphuric acid which precipitates
barium sulphate.
To identify silver further, dry the hydrochloric acid pre-
cipitate and fuse in a porcelain crucible with a little potassium
cyanide. Extract the melt with hot water. Metallic silver
remains undissolved.
To confirm the presence of lead, dissolve the hydrogen sul-
phide precipitate in hot nitric acid and evaporate the solution
to dryness. Dissolve the residue in water, filter and test the
solution for lead with sulphuric acid or potassium chromate.
To identify barium further, collect the sulphuric acid pre-
cipitate upon paper, thoioughly wash and test upon a clean
platinum wire in a non-luminous flame. Barium imparts a
yellowish green color to the flame. To avoid any mistake,
examine this flame with the spectroscope. These reactions
of identification are always necessary in toxicological analysis.
SYNOPSIS OF GROUP HI
Heat the residue from distillation of volatile poisons, or a
portion of original material, in a glass flask or porcelain dish
1 Even in the absence of barium, lead and silver, such a sediment nearly
always appears. In that case it usually consists of the material of the porcelain
crucible, the glazing of which is partially attacked in the fusion process.
2 Use 5-6 cc. of an acid prepared by mixing i volume of concentrated nitric
acid and 2 volumes of water.
172
DETECTION OF POISONS
upon the water-bath with dilute hydrochloric acid (12.5 per
cent.) and potassium chlorate and shake frequently or stir.
When most of the material is dissolved and the solution is
yellow, dilute with water. Add a few drops of sulphuric acid
and filter the cold solution.
Material. Treated with HC1 and KC1O 3 . Dilute H 2 SO 4 . Filter.
Filtrate. 1 Saturated warm with H 2 S.
Residue. Tested
for "Metallic
Poisons IV."
Ag, Pb, Ba.
Precipitate. Treated with hot
(H 4 Nj,S, and (H 4 N)OH.
Filtrate. Tested
for "Metallic
Poisons III."
Cr, Zn.
Filtrate. Tested
for "Metallic
Poisons I."
As, Sb, Sn, Cu.
Residue. Tested
for "Metallic
Poisons II."
Hg, Pb, Bi, Cu, Cd.
Action of Heavy Metals
Most salts of heavy metals, as lead, copper, mercury, silver, uranium and
bismuth, precipitate proteins. These compounds are metallic salts of albumins
(albuminates) . In combining with the oxides of these metals, proteins behave
like acids. If the metal albuminates first formed are insoluble, or only slightly
soluble in the body fluids, they are non-toxic or only slightly toxic. But a sol-
uble albuminate is transported throughout the organism and exerts a toxic action.
Every cell in contact with the dissolved metal may be poisoned. Mercury
albuminate is an example of the latter class of metal albuminates. Being sol-
uble in sodium chloride and protein solutions, it acts as a powerful poison.
Copper albuminate on the other hand is not appreciably soluble in solutions of
sodium chloride, hydrochloric acid or proteins. Not entering the circulation, it
is as good as non-toxic. Lead and silver albuminates are like copper albuminate
as regards solubility in the solvents mentioned. But if a heavy metal, which
forms a difficultly soluble albuminate, finds its way into the organism in organic
combination so that it cannot be precipitated by proteins, for example, copper in
union with tartaric acid, it then is as poisonous, or nearly as poisonous, as
1 If this filtrate contains much free hydrochloric acid, remove most of the
acid by evaporation. Then add ammonia until alkaline and finally acidify
with dilute nitric acid.
METALLIC POISONS 173
mercury in corrosive sublimate. Administration intravenously of 20 mg.
of such copper causes the death of an adult rabbit.
Consequently precipitation takes place wherever the salt of a heavy metal
comes in contact with proteins. The term corrosion is applied to such an oc-
currence. The,re is always present the metallic oxide, protein and the acid
originally combined with the metal. As a rule the acid is loosely held by the pre-
cipitate and is washed away by the circulating blood. The corrosive action of
salts of heavy metals is due both to the union of the metallic oxide with protein,
living protein being changed to dead metal albuminate, and to the caustic action
of the free acid. Therefore the intensity of the action of the salt of the heavy
metal depends upon the nature of the given metal albuminate. The degree of
solubility is especially important (see above), also the quantity and strength of
the free acid. Salts of heavy metals not only may affect the place of application
but they may give rise to serious changes where eliminated, as in the intestines
or kidneys. Before elimination they may also seriously harm parenchymatous
organs like the liver, as well as the circulatory organs. Finally salts of heavy
metals have an important action upon the blood. R. Kobert and his collabor-
ators have found that white as well as red blood-corpuscles may combine with
metals and thus act as antidotes. Kobert has shown that the substance of red
blood-corpuscles is capable of taking up a considerable quantity of a heavy metal.
A chemical compound (metal haemoglobin) is formed and the oxyhaemoglobin
spectrum is not changed. Thus lead speedily impairs the vitality of red blood-
corpuscles. Consequently red blood-corpuscles are killed in large quantity in
lead poisoning.
Fate, Distribution and Elimination of Metals in the Human Body
Arsenic. Elimination of arsenic takes place mainly through the urine. It
begins several hours (7-12) after administration and, after a single dose, usually
lasts 4-7 days. A great many experiments have shown the duration of arsenic
elimination in urine to vary from a few days to several weeks. Some observers
have found arsenic in urine 80, and even 90 days, after poisoning. Conse-
quently in suspected arsenic poisoning first examine the urine! In arsenic
poisoning the urine is usually diminished in volume and contains albumin and
blood-corpuscles.
As regards retention of arsenic by different organs, large quantities of the poison
are usually found in the liver. Examine also the stomach and intestines with
contents, since most of the poison will obviously be in these organs in case of
recent administration. The spleen, kidneys and muscles usually contain arsenic.
But the brain rarely shows more than traces of the poison. On the other hand,
the bones in many instances have contained arsenic. This fact has given rise to
the hypothesis that arsenic is capable of replacing phosphoric acid in the bones.
During the first stage in elimination of arsenic from the organism the poison
appears to be deposited in the bones as calcium arseniate. If large doses of
difficultly soluble arsenic compounds, as white arsenic, or small doses of soluble
compounds have been taken, the liver alone appears to arrest and retain the
poison. Probably the quite stable arseno-nucleins are formed in that organ.
From experiments where the organism was deluged with easily soluble arsenic
174 DETECTION OF POISONS
compounds, Chittenden concluded that the brain can arrest and retain the poison
and that arsenic then accumulates there. But after administration of white
arsenic or Schweinfurt green, only traces of the poison can be detected in the
brain. Two instances of arsenical poisoning are in favor of this view. In the
first case a woman died 9 hours after eating a highly arsenical soup. The liver
weighing 1259 grams, contained 76 mg. of arsenious oxide; the kidneys and
bladder 0.6 mg.; and half the brain only a faint trace of the poison. In the
second case a young woman died a day after taking Schweinfurt green. In
this instance too the brain contained only traces of the poison. In experiments
upon animals most of the arsenic was always found in the liver.
Normal Arsenic 1
The view that certain organs of the body may contain arsenic as an essential
constituent has led to the use of the term normal" to distinguish such arsenic
from that entering the organism in food or drugs. The introduction of this term
into chemical literature is unfortunate, because it suggests the possibility of two
kinds of arsenic. Such a notion has no foundation whatever. Arsenic is arsenic
and no test capable of showing more than one kind is known. A committee of
the French Academy of Sciences 2 after carefully investigating this matter came
to the conclusion that arsenic never occurs normally in the human body. But
within recent years A. Gautier 3 after making many analyses of different materials
has come to the opposite opinion. Gautier thus summarizes his results in one of
his papers: 4
"Speaking from a medico-legal point of view, I would state that arsenic,
aside from the thyroid, mammary and thymus glands, never cccurs in the human
body except in the skin, hair, bones, milk and sometimes in the faeces and then
only in traces which are often infinitesimal. Excepting the brain, the other organs
and fluids, especially those forming the bulk of the body, as muscular tissue, liver,
spleen, kidneys, lungs, blood, urine, etc., fail to show the slightest trace of arsenic
If a chemist therefore examines individually these arsenic-
free organs by my method or by one less delicate and finds traces, especially
appreciable traces, of this metalloid, such arsenic has been absorbed during life
either medicinally or criminally."
Gautier found the largest quantity of arsenic in the thyroid gland (0.75 mg.
in 100 grams of gland) but this result has not yet been confirmed. Other
chemists to be sure have found arsenic in the thyroid gland but in much smaller
quantity. These same chemists have also found arsenic in organs which Gautier
says are non-arsenical. The following is a summary of their results but the
original papers should be consulted for full details:
1 The brief account of "normal arsenic" in the German edition seems insuffi-
cient. After thoroughly examining the literature, the translator has therefore
decided to treat this subject more fully. Tr.
2 Comptes rendus de 1'Academie des Sciences 12, 1076-1109 (1841).
3 Ibid., 129, 929-936 (1899).
4 Ibid., 130, 284-291 (1900).
METALLIC POISONS
175
Observer
Material
Arsenic found
Remarks and
conclusions
Gautier 1
Human and ani-
0.75 mgr. in 100
Used 6 glands and
mal organs and
grams of human
assumed uniform
other material
thyroid gland
distribution of As
Bertrand 2
Only animal ma-
0.015 m S r - per 100
Concludes As is a
terial
grams of dried
normal constitu-
sponge
' ent of protoplasm
Schaefer 3
Human organs
0.007 mgr. per 100
Concludes As may
grams of human
occur in all organs
thyroid
but found many
free from As
Pagel*
Human and ani-
Positive but not
Found testes ar-
mal organs
quantitative
senical but Gautier
says they are not
On the contrary several chemists have carefully analyzed human and animal
organs, either finding no arsenic or detecting this metalloid in mere traces, which
are inconstant in occurrence and confined to no special organ. The table on
the opposite page briefly summarizes their results.
The results set forth in this table place "normal arsenic" in a doubtful posi-
tion at least. If it is a reality and not a fancy, the quantity of arsenic, compared
with that obtained in an analysis actually dealing with this metalloid, is so
minute that the toxicologist need feel no concern. If he has conducted his analy-
sis with every precaution as regards reagents and method and obtained a distinct
mirror, he may dismiss the "normal arsenic" chimera and accept the result as
due to arsenic that has entered the body from some external source. Kunkel 5
has summed up the matter in these words.
"The so-called normal arsenic, if there is such a thing, does not affect the
results of forensic chemistry, because the so-called normal quantities are so
exceedingly small (o.oi or even o.ooi mg. in an organ) that the quantities neces-
sary to furnish a satisfactory forensic proof, which are a hundred or even a thou-
sand times greater, must be regarded as an entirely different and much higher
order of magnitudes."
Antimony. Elimination of antimony takes place largely through the urine.
The rest of the metal is found chiefly in the liver and gastro-intestinal tract, as
1 Loc. cit.
2 Annales de 1'Institut Pasteur 17, i-io (1903); Comptes rendus de PAcademie
des Sciences 135, 800-812 (1902); and Bulletin de la Societe Chimique de Paris
(3), 27, 1233-1236 (1902).
3 Annales de Chimie Analytique 12, 52-58 and 97~ioi( 1907).
4 Dissertation, University of Nancy, 1900.
3 Zeitschrift fur physiologische Chemie 44, 511-529 (1905).
176
DETECTION OF POISONS
Observer
Material
Results
Conclusion
Hodlmoser 1
Human thyroid
20 analyses
Liver gives positive
gland and liver
Negative or few
results as often as
traces
thyroid gland
Cerny 2
Human and ani-
28 analyses
Minute traces may
mal thyroid and
Negative or faint
appear but not
thymus glands.
traces
constant
Human liver
Ziemke 3
Various human
Over 40 analyses.
Not a normal con-
organs
Negative. One
stituent of human
case doubtful
organs
Wieser*
Various human
32 analyses. Mostly
Traces inconstant
and animal or-
negative but few
and due to chance
gans
traces
contamination
Kunkel 5
Various human
Negative
No such thing as
and animal or-
normal As
gans
Bloemendal 6
Various human
Mostly negative
No such thing as
and animal or-
but few traces
normal As
gans
Warren 7
Human thyroid
32 analyses. Nega-
No such thing as
gland
tive except two
normal As
slight traces
well as in the kidneys and brain. Pouchet found antimony in bones, skin, hair
and chiefly in the intestinal tract. A large part of the ingested poison may be
eliminated by emesis. A part is apparently retained in the body for some time,
since antimony has been detected in the liver and in the bones months after the
last administration.
Lead. Lead is eliminated in urine and faeces. Elimination by the faces
always exceeds that by the Urine, even when the lead has not been taken by
the mouth. Mann" (see R. Robert, Intoxikationen), for example, in the case
of two patients was never able to find more than 0.6 milligram of lead in the
urine collected during 24 hours, whereas the faces during the same period con-
tained 2-3 milligrams of lead. In lead poisoning the metal has been found in the
^eitschrift fur physiologische Chemie 33, 329-344 (1901).
2 Ibid., 34, 408-416 (1902).
3 Vierteljahrsschrift fur gerichtliche Medizin (3) 23, 51-60 (1902).
' Dissertation, University of Wtirzburg (1903).
* Zeitschrift fur physiologische Chemie 44, 511-529 (1905).
6 Dissertation, University of Leyden (1908).
7 W. H. Warren, analyses not published.
8 Zeitschrift fur physiologische Chemie 6, 6 (1882).
METALLIC POISONS 177
saliva, bile and in both red and white blood-corpuscles. In animals relatively
most of the lead has been found in the kidneys, after which come bones, liver,
testes and finally the brain and blood. In experiments with sheep Ulenberger
and Hofmeister obtained the following results:
Organs and fluids Pb in grams per 1000
grams
Kidneys 0.44-0,47
Liver 0.3 -0.6
Pancreas o . 54
Salivary glands 0.42
Bile 0.11-0.40
Bones 0.32
Faeces ' 0.22
Spleen 0.14
Blood 0.050.12
Urine 0.06-0.08
Lead is eliminated especially by the bile and in acute poisoning this secretion
may contain more lead than any of the other organs or secretions. Oliver 1 has
given the following results for human material:
Organs Pb in grams per 1000
grams
Liver 0.0416
Spleen 0.039
Large brain 0.0216
Small brain 0.0086
Kidneys 0.013
Heart 0.0005
Elimination of lead by the urine is said, in the case of man, not always to'be
uniform. The urine is free from lead for a long time and later, without further
administration, again contains the metal. This behavior is in harmony with the
fact that lead can be retained in the organism many months. In chronic lead
poisoning the brain has frequently been found to contain much lead. In this
kind of poisoning elimination of the metal is always more abundant by the faeces
than by the urine.
Chromium. Chromic acid and soluble chromates and dichromates are quite
toxic. The mucous membranes absorb alkaline chromates rapidly and severe,
acute poisoning occurs. The poison causes intense pain in the stomach and
intestines, collapse and kidney derangement which may terminate fatally in a
few hours. Other symptoms are nausea, vomiting of yellow matter which later
is tinged with blood, diarrhoea and even bloody stools, intense thirst, emaciation,
great anxiety, severe pain in the abdomen, faint and quickened pulse "the
cholera picture." (Kunkel, Toxikologie.) The statements regarding the quan-
tity of an alkaline chromate, capable of producing acute poisoning, agree fairly
well. Even a few decigrams (0.2 gram) may cause very serious symptoms which
1 The Lancet, March, 1891.
12
178 DETECTION OF POISONS
sometimes terminate fatally. Chromic acid is eliminated mainly by the urine but
partly by the intestines. Elimination takes place rapidly and the body is soon
free of the poison. Four days after administration of quite large quantities of a
chromate, the urine and faeces are said to contain only traces of this metal.
Copper. Only a small amount of copper, varying with different compounds, is
absorbed by the intestines and carried into the circulation. Sodium cupric
tartrate and copper salts of fatty acids are absorbed most easily. Copper poison-
ing rarely occurs from introducing a copper compound into the stomach. Copper
compounds in large amounts act as local caustics and occasion severe pain in the
stomach. Vomiting and the sense of taste make it impossible to take much of a
copper compound. Foods containing copper are unpalatable. The sense of
taste as well as after-taste prevent one from swallowing such in any quantity.
Food, containing 0.5 gram of copper per kilogram, has a marked taste. Irresisti-
ble nausea, steadily increasing, soon makes it impossible to take more of the food
containing copper. Elimination of copper by the urine is very sb'ght. Copper
absorbed from the intestines is arrested by the liver where it accumulates.
Traces of copper have frequently been found in the human liver. The author in
toxicological analyses has repeatedly found weighable quantities of copper in
the livers of adults who had not taken copper salts beforehand, except possibly
for suicidal purposes. The liver is the most important organ for the detection of
copper, next to which come the bile, kidneys and the gastro-intestinal mucosa.
Copper is said to be in the liver as a nuclein compound. In the case of blood,
copper is located not in the serum but in the corpuscles.
Mercury. Distribution of mercury in the body is said to be always the same,
no matter what the method of administration is. It is immaterial whether it is
introduced by the mouth, hypodermically or from an abrasion. Elimination of
mercury takes place through the saliva, sweat, bile, gastro-intestinal mucosa and
urine. Elimination in the saliva seems to be constant, since mercury can always
be detected in the saliva during use of mercurials in lues. A relatively large
quantity of this metal is said to be eliminated in the sweat. Opinions differ as to
the relative quantities eliminated by the urine and intestines. Usually elimina-
tion by the intestines exceeds that by the kidneys. Recent experiments appear
to show that mercury is eliminated in the urine regularly and in slowly increasing
quantity and then slowly diminishes. Elimination of mercury ceases after 6-9
months and even later. In a most favorable case the total quantity of mercury
eliminated in the urine amounts to about 50 per cent, of the total quantity taken
but is frequently much less. In mercury poisoning the kidneys, of all the organs
persistently contain most of the poison even for weeks. Then follow the liver,
spleen, bile and intestinal mucosa. In toxicological analysis the urine should
also be examined, though in acute poisoning it always contains only a fraction of
a milligram of mercury in a liter. In severe mercurial poisoning the metal may
be said to occur in all organs and secretions.
Electrolytic Separation of Mercury from Urine
Heat a liter of urine upon the water-bath about 2 hours with 5-6 grams of
potassium chlorate and 10 cc. of concentrated hydrochloric acid and shake fre-
quently. Evaporate to 300 cc. and use this solution for the electrolytic separa-
METALLIC POISONS 179
tion of mercury. Use a Bunsen battery of 3-4 cells, or any other galvanic ap-
paratus having the same strength of current. A thin sheet or rod of gold 2 mm.
thick and 6-10 cm. long serves as the cathode, and a piece of platinum wire of
about the same thickness as the anode. Place the electrodes in the solution 2-4
cm. apart and allow the experiment to run 24-48 hours. Wash and dry the gold
cathode, upon which the mercury is deposited, and place in a glass tube (20 cm.
long and 4-5 cm. in diameter). This tube is sealed at the bottom and reduced
at the top to smaller size. Apply heat until all the mercury is expelled from the
gold. Deposit the sublimate 3-4 cm. beyond the top of the gold rod. Then seal
the tube below the sublimate. Introduce a small crystal of iodine into the tube
and seal the other end. Heat the iodine carefully over a small flame to bring it
into contact with the mercury. The two elements combine to form red mercuric
iodide.
Sometimes it is convenient to precipitate mercury from urine upon other
metals, for example, copper, gold, brass and zinc dust. Witz heats to boiling
with hydrochloric acid and concentrated potassium permanganate solution to
destroy organic matter. Use 10 cc. of concentrated hydrochloric acid and 15-20
cc. of potassium permanganate solution for 500 cc. of urine. Slowly pass the
decolorized liquid through a glass tube over a copper spiral. Wash the copper
with potassium hydroxide solution, then with absolute alcohol and cleanse with
filter paper. Finally dry at 70-80 and heat in a glass tube. Treat the mercury
sublimate with iodine as described. Satisfactory deposition of mercury upon
other metals (gold, copper) requires previous destruction of organic matter in the
urine by hydrochloric acid and potassium chlorate. Otherwise, organic sub-
stances deposited upon the metal interfere with the iodine test for mercury.
Silver. In severe poisoning silver has been found in bile, faeces and in many
organs. In acute poisoning the urine is usually free from silver, whereas the
contents of the intestines may contain the metal even after subcutaneous injec-
tion. Absorbed silver salts appear to be reduced in all parts of the body. Ex-
amination of the bodies of persons who have suffered from argyria (chronic silver
poisoning) has disclosed precipitation of metallic silver in the organs. Silver
salts, added to albumin solutions, form very stable compounds usually amor-
phous. Silver may evince even greater affinity for albumin than for chlorine.
All observers agree that only very little of the silver reaching the intestines is
absorbed. After silver medication the stools are black from silver sulphide.
Frequent quantitative estimations of silver in argyrotic organs have been made.
In one case the liver gave 0.047 per cent, of silver and the kidneys 0.061 per cent.
In the condition called argyria, or argyrosis, the skin is black. Internal adminis-
tration of silver salts causes this color to develop gradually. By' degrees it may
become quite marked even causing disfigurement.
Quantitative Estimation of Silver in Organs and Urine
V. Lehmann, 1 in determining silver in organs (liver, kidneys, spleen, brain),
first thoroughly dries the finely ground material and then fuses with sodium
carbonate and potassium nitrate. Extract the melt with water and dissolve
the insoluble residue of metallic silver in hot nitric acid. Evaporate this solution
1 Zeitschrift fur physiologische Chemie 6, 19 (1882).
180 DETECTION OF POISONS
to dryness upon the water-bath and precipitate silver as chloride. Avoid a
large excess of hydrochloric acid.
Mix urine with sodium carbonate and potassium nitrate, evaporate to dryness,
fuse the residue and treat the melt as described.
Uranium. Experiments made by R. Robert have shown that uranium,
administered subcutaneously or intravenously, is the most toxic of all metals.
Uranyl acetate is an excellent precipitant of albumins and the other uranyl salts
must behave in much the same way. Consequently internal administration
of concentrated solutions of uranyl salts destroys the mucous surfaces they
touch, for example, that of the stomach, changing the living stomach wall to
dead uranyl-albuminate. Uranyl salts therefore must be classed among the
powerful caustic poisons. In addition to acting as local corrosives, uranium
salts resemble hydrocyanic acid in partially arresting internal oxidation in the
organs and occasioning the severest disturbances of metabolism.
Bismuth. This metal becomes quite toxic when it reaches the blood. Bis-
muth solutions, prepared by dissolving bismuthous hydroxide in tartaric or citric
acid and then neutralizing with sodium or ammonium hydroxide solution, have
been repeatedly administered to animals subcutaneously and intravenously.
The smallest lethal dose of these double bismuth salts, injected subcutaneously,
was found to be but 6 mg. per kilogram for a dog or cat and 24 mg. per kilo-
gram for a rabbit. Bismuth salts insoluble in water produce entirely differ-
ent results when administered internally. Bismuth subnitrate and similar
salts dissolve very slightly in the highly diluted hydrochloric acid of the gastric
juice. Consequently very little bismuth is conveyed to the blood. Most of the
bismuth taken by the mouth reaches the intestines. Instead of being absorbed,
it is changed to bismuthous sulphide by the hydrogen sulphide always present.
Absorbed bismuth is eliminated by the saliva, bile, urine, mucous lining of the
mouth, stomach, small and large intestine 'and also the milk. If an animal is
poisoned by bismuth, the metal can be detected in the urine, bile, liver, kidneys,
spleen, walls of the intestines as well as in the bones. Different observers have
found the metal in especially large amount in the milk but very little in the kid-
neys and liver.
Zinc. There is no doubt that zinc salts reaching the intestinal tract are
absorbed in very small quantity. As yet there is no satisfactory explanation of
the fate of absorbed zinc. In zinc poisoning large amounts of the metal have
been found repeatedly in the liver and bile. This may mean that zinc is arrested
by the liver and eliminated in the bile. Lehmann 1 after 335 days killed a dog
that had been fed for a considerable time upon zinc carbonate. The following
organs, arranged according to the quantity of metal in each, contained zinc:
liver, bile, large intestine, thyroid gland, spleen, pancreas, urine, kidneys, blad-
der, muscle, brain, lymphatic gland, stomach, small intestine, lungs, blood. Oc-
casionally considerable quantities of zinc may be taken with articles of food and
drink. All acids dissolve metallic zinc very freely. Even water containing
carbon dioxide is a solvent. Consequently it may be in drinking water from gal-
vanized pipes. All kinds of food and drink, kept in zinc vessels or vessels
coated with zinc, may contain more or less of this metal. Plants grown upon
1 Archiv fur Hygiene 28, 291 (1896).
METALLIC POISONS 181
soil containing zinc take up the metal. Zinc has also been found repeatedly in
parts of human cadavers under circumstances precluding all possibility of poi-
soning by this metal. Even considerable quantities of zinc have been found
in the human liver.
Tin. The cases of tin poisoning thus far observed resemble those of copper
and zinc. What knowledge there is regarding the toxic action of absorbed tin
has been gained from experiments upon animals. These experiments show that
small quantities of tin are absorbed and eliminated in the urine, when ordinary
tin compounds are brought into the stomach. But thus far distinct symptoms
of poisoning by such quantities of the metal have not been confirmed. (Kunkel,
Toxikologie.)
White 1 failed to produce poisoning by bringing tin into a dog's stomach. The
animal received sodium stannous tartrate in increasing doses for 22 days, the
daily amount being 0.02-0.06 gram. Yet the animal absorbed tin. In the urine,
during an experiment lasting 8 days, White found 0.02 gram of tin. But the
tin salt mentioned, introduced directly into the circulation of the animal, was
quite toxic in its action. Stannous chloride, administered for a very long time
to a dog, produced symptoms of poisoning. The urine in this case contained
small quantities of tin. Kunkel (Toxikologie) states that tin has a very slight
poisonous action. Apparently it is eliminated very rapidly by the kidneys.
Quite probably this prevents accumulation of the metal in the body and conse-
quent poisoning. The fact that White did not observe toxic symptoms, after
feeding a dog for 22 days with relatively large quantities of easily absorbable
sodium stannous tartrate; and that Ungar and Bodlander 2 failed to produce
derangements with the same compound, until it had been administered for a year,
prove that tin is quite free from toxic properties. Hence, tin vessels may be used
and preserved articles of food containing tin have practically no deleterious
action upon health.
J Archiv fur experimentelle Pathologic und Pharmakologie 13, 53.
2 Zeitschrift fur Hygiene, 2, 241.
CHAPTER IV
POISONS NOT IN THE THREE MAIN GROUPS
MINERAL ACIDS
Hydrochloric, Nitric and Sulphuric Acids
To detect free mineral acid, extract a portion of material with
cold water, filter and test as follows, if the solution is strongly
acid:
1. Methyl Violet Test. Add a few drops of an aqueous
(o.i : 1000) or alcoholic (i : 100) solution of methyl violet 1 to
a small portion of filtrate. A free mineral acid produces a blue
or green color.
2. Methyl Orange Test. Add a few drops of a dilute aqueous
solution of methyl orange 2 to the filtrate. A red color indicates
free mineral acid.
3. Congo Paper Test. Even very dilute solutions of free
mineral acids turn "Congo paper" blue.
4. Giinzburg's Test. Mix a few drops of the filtrate with
3-4 drops of Giinzburg's reagent 3 and evaporate to complete
dryness upon the water-bath, or over a small flame. Fre'e
hydrochloric or sulphuric acid gives a fine red or reddish yellow
residue. Nitric acid gives more of a yellowish red residue.
1 Methyl violet is the hydrochloride of hexa-methyl-para-rosaniline :
(CH 3 ) 2 N.C 6 H 4V /CH = CH\ V
>C = C >C = N(CH 3 ) 2 C1 (quinoidal form)
(CH 3 ) 2 N.C,H/ \CH = CH/
or
V
(CH 3 ) 2 N.C 6 H 4X /C6H 4 .N(CH 3 ) 2 C1
(CH 3 ) 2 N.C 6 H 4 '
2 Methyl orange = Dimethyl-amino-azobenzene-4-sulphonic acid:
(CH 3 ) 2 N. C 6 H 4 .N = N. C 6 H 4 . S0 2 OH.
The sodium salt of this sulphonic acid also appears in commerce under the name
"methyl orange."
3 See page 321 for the preparation of this reagent.
182
POISONS NOT IN THE THREE MAIN GROUPS 183
5. Sulphocyanate Test. Add a little potassium sulphocya-
nate solution to ferric acetate solution and dilute with water
until yellow. Then add the solution to be tested. Free mineral
acid produces a blood-red color. Traces of free mineral acid,
especially if considerably diluted, do not give a red color until
several minutes have elapsed.
One or more of these general tests, which furnish evidence of a
free mineral acid, must always accompany the special tests to
be described later. Not only free mineral acids give the special
tests but in certain cases their salts. Chlorides, sulphates and
nitrates are normal constituents of nearly all vegetable and
animal materials. As a rule an examination of cadaveric
material for mineral acids is necessary only when the autopsy
points conclusively to such poisoning. That is to say, when
there are characteristic corrosions and discolorations about the
face, mouth, cesophagus and stomach. If general tests show the
presence of free mineral acid, make special tests for the particu-
lar acid.
Hydrochloric Acid
1. Chlorine Test. Warm a little of the aqueous extract, if
not too dilute, with finely powdered manganese dioxide. Free
hydrochloric acid yields chlorine, recognized by its color and
odor, or by passing the gas into potassium iodide solution and
liberating iodine. Hydrochloric acid exclusively does not give
this test. A chloride (NaCl) and free sulphuric acid give chlor-
ine under the same conditions.
2. Distillation. If possible, separate hydrochloric acid from
other substances by distillation. The concentration of the
acid is especially important, since dilute hydrochloric acid upon
distillation at first yields only water. Hydrochloric acid 1
itself does not begin to distil until the concentration is about
10 per cent. Since a dilute hydrochloric acid is usually ex-
amined, distil the material mixed with water, or preferably a
1 In the distillation of 100 cc. of i per cent, hydrochloric acid, the first 90 cc.
of distillate will contain only traces of hydrochloric acid, whereas the last portion
will contain most of the acid.
184 DETECTION OF POISONS
filtered aqueous extract, nearly to dryness. In such a dis-
tillation apply heat by means of an oil-bath. To detect hydro-
chloric acid in the distillate, acidify with dilute nitric acid and
add silver nitrate solution. Frequently a quantitative estima-
tion of hydrochloric acid is required. In the absence of other
acids, titrate the distillate with o.i n-potassium hydroxide
solution, using phenolphthalein as indicator. Otherwise, esti-
mate the acid gravimetrically, precipitating with silver nitrate
and weighing silver chloride, or volumetrically by Volhard's
method. In the latter case, precipitate hydrochloric acid with
o.i n-silver nitrate solution in excess and subsequently estimate
that excess by titration with o.i n-ammonium sulphocyanate
solution, using ferric alum as indicator. Since the human
stomach normally contains 0.1-0.6 per cent, of free hydro-
chloric acid, an examination of stomach contents for this acid
must always include a quantitative estimation.
Nitric Acid
The human body normally contains only a very small amount of nitrates.
When present, they are due usually to vegetable foods which contain small
quantities of nitrates. Human urine almost always shows traces of the salts of
nitric and nitrous acids. The chemical examination of cadaveric material need
not include tests for nitric acid, unless the autopsy affords evidence of poisoning
by this acid, as distinct signs of corrosion about the lips, mouth, oesophagus and
stomach and sometimes perforation. These parts are more or less yellow or
yellowish brown. A yellow froth is said to exude from the mouth and nose of
the cadaver. Also the stomach contents are yellow in concentrated nitric acid
poisoning. If the concentration of the acid is less than 20 per cent., these specific
changes may not appear in the gastro-intestinal tract. Nitric acid taken inter-
nally, dilute or concentrated, appears at once in the urine.
Detection of Nitric Acid
i. Distillation. If possible, extract the material direct with
water, filter and test the filtrate for nitric acid in the usual way.
When the quantity of nitric acid is large, separate it from other
substances by distilling the filtered aqueous extract. Apply
heat by means of an oil-bath. Nitric acid 1 does not distil, until
1 If ioo cc. of i per cent, nitric acid are mixed with bread crumbs and distilled,
most of the acid will be in the final 10 cc. of distillate.
POISONS NOT IN THE THREE MAIN GROUPS 185
it reaches a definite concentration. At the same time a large
part of the acid combines with organic substances, if any are
present, forming nitro-derivatives, xanthoproteic acid, etc.
Nitric acid may also cause oxidation. Consequently the dis-
tillate does not contain all the acid originally present. The
residue from such a distillation is usually distinctly yellow.
Toward the end of distillation brown vapors of nitrogen peroxide
often appear. Such a distillate, added to starch paste and po-
tassium iodide, produces an immediate blue color in presence of
dilute sulphuric acid.
To detect nitric acid in the distillate, employ the following
tests:
2. G. Fleury's Procedure. 1 Extract the finely divided
material with absolute alcohol, filter and add slaked lime in
excess to the filtrate. To decompose any nitric acid ester
present, let the mixture stand 12 hours, filter and evaporate the
filtrate to dryness. Dissolve the residue in 95 per cent,
alcohol, expel alcohol from the filtered solution and finally
test an aqueous solution of the residue for nitric acid.
Fleury has obtained by means of this method about 20 per
cent, of the nitric acid from animal material. This procedure
converts the acid into its calcium salt which is soluble in
alcohol. But sodium nitrate is also quite soluble in 95 per
cent, alcohol (i : 50). Therefore, if the final residue gives a
faint test for nitric acid, the proof of free acid in the original
material is not conclusive. The following method obviates this
difficulty.
3. Baumert's Procedure. 2 Neutralize the material itself, or
its aqueous extract, with milk of lime, dry and extract with
alcohol. Or, after neutralization with milk of lime or calcium
carbonate, evaporate to a syrup, stir and mix with alcohol.
Distil the filtered alcoholic extract obtained in either way,
dissolve the residue in water, filter and evaporate the solution.
Dissolve the residue again in alcohol and allow this solution
to stand for several hours in a closed flask with about the
1 Annales de Chimie analytique appliqu^e 6, 12.
2 Lehrbuch der gerichtlichen Chemie, second edition (1907).
186 DETECTION OF POISONS
same volume of ether. Filter this alcohol-ether solution, evapo-
rate the solvent and dissolve the residue in a little water.
Apply the following nitric acid tests to this solution:
(a) Diphenylamine and Sulphuric Acid Test Blue color.
Add a few drops of diphenylamine sulphate solution 1 to the
aqueous extract, or distillate, and carefully pour this mixture
upon pure concentrated sulphuric acid free from nitric acid.
If nitric acid is present, a blue zone appears where the two
liquids meet.
(b) Brucine and Sulphuric Acid Test. Red color.
Mix the liquid to be tested with the same volume of brucine
sulphate solution 2 and carefully pour this mixture upon pure
concentrated sulphuric acid. If nitric acid is present, a red
zone appears where the two liquids meet.
(c) Ferrous Sulphate and Sulphuric Acid Test. Saturate the
liquid to be tested with pure ferrous sulphate and carefully
pour this solution upon pure concentrated sulphuric acid. If
nitric acid is present, a black zone appears where the two liquids
meet.
(d) Copper Test. Place a small piece of clean copper (wire
or sheet) in nitric acid and heat. Red-brown vapors of nitrogen
peroxide (NO 2 ) appear.
Sulphuric Acid
Nearly all animal and vegetable substances normally contain sulphates. Con-
sequently an examination for free sulphuric acid must exclude its salts. There
is no need of examining cadaveric material for the free acid, unless marked corro-
sion and discoloration of lips, mouth, oesophagus and stomach indicate its pres-
ence. There are eschars upon the lips and the mucous lining of the mouth is
grayish white. The white coating on the back of the tongue may have been
dissolved exposing the firm, brownish muscular tissue beneath. The tongue
often looks as if it had been boiled. The mucous lining of the oesophagus is
1 Prepare this solution by dissolving i gram of diphenylamine,
in 5 grams of dilute sulphuric acid and 100 cc. of water.
2 Prepare this solution by dissolving i gram of brucine in 5 grams of dilute
sulphuric acid and 100 cc. of water. The sulphuric acid used must give none of
the tests for nitric acid. If it does not meet this requirement, heat in a platinum
dish to expel interfering nitrous substances. Or distil the acid from a small
retort, rejecting the first part of the distillate.
POISONS NOT IN THE THREE MAIN GROUPS 187
much wrinkled and coated gray. Externally the stomach is usually brown or
slate-gray and its contents black. Frequently in sulphuric acid poisoning there is
perforation of the stomach wall and brownish black masses find their way into
the abdominal cavity. There may be black spots in the stomach, due according
to R. "Kobert (Intoxikationen) not to charring, as previously supposed, but to
brown-black haematin. Acids decompose the blood-pigment oxyhaemoglobin
mainly into haematin and protein (globulin). Methaemoglobin and hsematopor-
phyrin may also be formed. Acids produce the latter from haematin and in the
change there is loss of iron. All three of these decomposition products of the
red blood-pigment, namely, methaemoglobin, haematin and haematoporphyrin
may be formed successively and then appear in the urine. The blood in the
stomach wall,s is often acid and then contains chiefly methaemoglobin and haema-
tin. The mucosa of the intestines even far down may be grayish white and
strongly acid.
Detection of Sulphuric Acid
1. Extract the finely divided material, if strongly acid, with
cold absolute alcohol and after some time filter. The solution
contains sulphuric acid but not sulphates. Evaporate the
alcoholic filtrate upon the water-bath, or, if the volume is large,
distil the alcohol. Dissolve the residue in a little water (10 cc.)
and heat the solution to boiling to saponify 1 ethyl sulphuric
acid. Filter and test the filtrate with barium chloride or lead
acetate solution. To prove that the precipitate is a sulphate,
mix with sodium carbonate and fuse upon charcoal. The
sodium sulphide formed blackens metallic silver in presence of
water, or gives hydrogen sulphide with acids.
2. Extract the finely divided material with water and apply
the following tests to the filtrate:
(a) Sugar Test. Evaporate some of the filtered extract in a
porcelain dish with a small particle of sugar. Free sulphuric
acid produces a black, carbonaceous residue.
(b) Sulphur Dioxide Test. Concentrate the filtered extract
upon the water-bath and heat in a test-tube with a few pieces of
copper. Free sulphuric acid generates sulphur dioxide, recog-
nized by its stifling odor. Distil the sulphur dioxide (preferably
in an atmosphere of carbon dioxide) into a little water and test
the distillate as follows:
1 HO.SO 2 .OC 2 H 8 + H 2 O = C 2 H 3 .OH + H 2 SO 4 .
188 DETECTION OF POISONS
a. Warm some of the liquid with a little stannous chloride
solution. A yellow precipitate of stannic sulphide 1 appears.
|8. Add iodo-potassium iodide solution drop by drop. The
color of the iodine disappears and at the same time sulphuric
acid is formed:
H 2 S0 3 + H 2 + I, = H 2 S0 4 + zHI.
Barium chloride then precipitates barium sulphate insoluble
in dilute hydrochloric acid.
To estimate sulphuric acid quantitatively, either precipitate
and weigh barium sulphate in the usual way, or titrate with
o.i n-potassium hydroxide solution, using phenolphthalein as
indicator.
1000 cc. of o.i n-potassium hydroxide solution = o.i gram-
equivalent of sulphuric acid = 4.0 grams of H 2 SO4.
Detection of Sulphurous Acid
Sulphur dioxide acts most injuriously when inhaled. It is very irritating to
the respiratory organs and also changes the blood-pigment. After death the
respiratory organs are found to be profoundly altered as when acted upon by
strong mineral acids. After severe poisoning by vapors containing sulphur
dioxide, the blood is dirty brownish red 2 and usually gives the haematin spectrum.
Human beings experience discomfort, if there are 0.015-0.02 volumes of sulphur
dioxide per 1000 volumes of air. Many persons become quite ill in a few minutes,
when there are 0.03 volumes of sulphur dioxide in 1000 volumes of air. The gas
produces a sharp, stinging sensation in the nostrils, sneezing and coughing. In
experiments upon mice, rabbits and guinea-pigs, Lehmann observed marked
toxic symptoms from air containing 0.04 volume per cent, of sulphur dioxide;
death ensued in 6 hours from 0.06 per cent.; and in 20 minutes from 0.08 per cent.
Articles of food and drink, preserved by means of sulphurous acid or its salts,
may injure the health, causing especially gastro-intestinal catarrh and other
chronic derangements. For this reason it is prohibited to preserve articles of
food and drink by means of sulphurous acid, sulphites and hyposulphites.
If the quantity of sulphur dioxide in air is not too small, its presence may be
recognized by its characteristic stifling odor. A strip of paper, moistened with
1 Sulphurous acid and sodium sulphite, added to stannous chloride solution
not too strongly acid, precipitate stannous sulphite, SnSO 3 , white and readily
soluble in hydrochloric acid. Warmed in presence of hydrochloric acid, sulphur
dioxide acts upon a stannous salt as an oxidizing agent. A precipitate of SnOioS 2
is formed, or H 2 S is evolved and SnCl 4 formed, depending upon the amount of
hydrochloric acid present. (Prescott and Johnson, Qualitative Chemical Analy-
sis. Fifth edition, page 86.)
2 Neutral sulphites cause the blood to become brick-red.
POISONS NOT IN THE THREE MAIN GROUPS 189
a solution of pure potassium iodate (KIO 3 ) and starch, turns blue in air contain-
ing sulphur dioxide owing to the formation of a compound of iodine and starch.
This reaction serves as a preliminary test for the detection of sulphurous acid and
hyposulphites in chopped meat, sausage meat and other meat products. Shake
the meat in an Erlenmeyer flask with phosphoric acid, suspend in the neck of the
flask from the stopper (see Fig. i, page 3) a paper strip prepared as described
and heat the flask upon the water-bath. The paper should not turn blue.
Explanation. Sulphur dioxide reduces potassium iodate (a) . Sulphuric acid
thus formed liberates hydriodic and iodic acids from their salts (/3 and y). The
iodine set free by the interaction of these two acids (5) finally turns the starch
blue.
(a) KI0 3 + 3 H 2 S0 3 = KI + 3 H 2 S0 4 ,
(0) 2KI + H 2 S0 4 = 2HI + K 2 S0 4 ,
(7) 2KI0 3 + H 2 SO 4 = 2HI0 3 + K 2 SO 4 ,
(6) HIO 3 + 5HI = 312 + 3H 2 O.
The official directions 1 for the detection and quantitative estimation of sulphur
dioxide in meat are as follows. Mix 30 grams of finely chopped meat with 200
cc. of boiled water in a 500 cc. distilling flask. 2 Add sodium carbonate solution
until the reaction is faintly alkaline.
Let the mixture stand for an hour and then completely expel air from the
apparatus by passing carbon dioxide through the tube extending to the bottom of
the flask. Then introduce into the Peligot tube (see below) 50 cc. of iodine
solution (5 grams of pure iodine and 7.5 grams of potassium iodide in a liter
of water). Raise the stopper of the distilling flask and, without stopping the flow
of carbon dioxide, add 10 cc. of 10 per cent, phosphoric acid solution. Then
carefully heat the contents of the flask and distil half the liquid, maintaining all
the while a current of carbon dioxide. Transfer the contents of the Peligot
tube, which should be brown, to a beaker, rinsing it out with water to prevent
loss of solution. Add a little hydrochloric acid, heat and by means of barium
chloride solution completely precipitate the sulphuric acid formed from the oxida-
tion of sulphurous acid by iodine.
H 2 S0 3 + H 2 O + I, = H 2 SO 4 + 2HI.
If this test is positive, then the meat examined contains either free sulphurous
acid, sulphites or hyposulphites. In the quantitative estimation the barium sul-
phate should be weighed in the usual manner.
OXALIC ACID
Oxalic acid and it s salts, for example, salt of sorrel, are quite toxic substances.
Administration of oxalic acid has terminated fatally in the case of adults in a few
1 Measures for putting into effect the law of the German Empire of June 3,
1900, relating to the inspection of beef-cattle and meats.
2 The apparatus prescribed for official examinations is a distilling flask, having
a capacity of 400-500 cc. and provided with a two-hole stopper for two glass
tubes entering the flask. One tube extends to the bottom of the flask and the
other only into the neck. The latter is connected with a Liebig condenser to
which a Peligot tube is fastened at the other end by a tight stopper.
190 DETECTION OF POISONS
minutes. Oxalic acid is very abundant in the vegetable kingdom in the form of
its acid potassium salt, KHC2O4, and calcium salt. Sorrel, wood-sorrel and
rhubarb are especially rich in salts of oxalic acid. Hence this acid may find
access to the body through food and drugs of vegetable origin. Moreover,
oxalic acid is a normal constituent in small quantity of human urine, 2-6 milli-
grams being excreted in the course of a day. Consequently in examining animal
material it is often necessary to supplement a positive qualitative test by a
quantitative estimation of oxalic acid.
Toxic Action. An important difference between mineral acids and oxalic
acid is the toxicity of salts of the latter. Not only do free oxalic acid and its
acid potassium salt, salt of sorrel, show poisonous properties but even very dilute
solutions of neutral sodium oxalate, Na2C2C>4, act in the same way. Therefore
in oxalic acid poisoning it is necessary to distinguish between local corrosion,
occurring at the point of application and also in part upon elimination, and re-
mote action due to absorption. Local action at the point of application is cor-
rosive like that of all acids. Local action at the place of elimination depends
upon the formation and insolubility of calcium oxalate. On account of the ease
wjth which the organism takes up oxalic acid and its alkali salts, the action or the
absorbed poison is rapid. The effects caused by its presence may be attributed
'to the fact that this acid removes in part from organs, as the heart, and from
body fluids (blood) the calcium they' require for their life processes, converting
it in part into insoluble calcium oxalate. Oxalates diminish the coagulating
power as well as the alkalinity of blood. On the other hand they increase the
quantity of sugar in the blood. In oxalic acid poisoning there is a depression
of the entire metabolism. This is also the case as regards taking up oxygen and
giving off carbon dioxide. The body temperature falls as the processes of metabo-
lism are retarded. Owing to withdrawal of calcium from the heart, that
organ is weakened and finally paralyzed. Local action upon the kidneys is due
to clogging of the injured urinary tubules by deposits of calcium oxalate. The
flow of urine may wholly cease in consequence of total impairment of the urinary
tubules and death may ensue from anuria and uraemia. Fatal poisonings from
large doses of oxalic acid are usually of short duration. R. Robert (Intoxika-
tionen) describes a case where death occurred within 10 minutes.
Bischoff 1 has made statements with regard to the distribution of oxalic acid in
the different organs of persons poisoned by this substance. In a case, which ter-
minated fatally in less than 15 minutes, the quantity of oxalic acid in each organ
was determined separately and found to be:
Weight Organ Oxalic Acid
2240 grams Stomach, oesophagus, intestine and
contents 2.28 grams.
7 70 grams Liver 0.285 grams.
290 grams Kidneys 0.0145 grams.
180 grams Blood from the heart 0.0435 grams.
40 grams Urine 0.0076 grams.
1 Berichte der Deutschen chemischen Gesellschaft, 16, 1350 (1883).
POISONS NOT IN THE THREE MAIN GROUPS 191
The quantity of oxalic acid in the liver is noticeably large. The kidneys and
Urine contain only a little of the poison, owing to the short duration of life after
poisoning. A striking thing about the urine excreted during oxalic acid poisoning
is the abundant deposition of crystallized calcium oxalate.
Detection of Oxalic Acid
To detect oxalate without discriminating between the free
acid, acid potassium salt or calcium oxalate, employ the follow-
ing method :
Add to the finely divided material 3-4 volumes of alcohol
and acidify with dilute hydrochloric acid. Stir frequently and
let the mixture digest 1-2 hours cold. Then filter through a
plaited paper moistened with alcohol and wash the residue with
alcohol. To prevent formation of ethyl oxalate during evapo-
ration, add about 20 cc. of water to the total filtrate. Evapo-
rate upon the water-bath until all alcohol is expelled. Pass
the aqueous residue through a small filter. Extract the filtrate
in a separating funnel 3-4 times with 5060 cc. portions of ether.
Let the total ether extract stand for
some time in a dry flask, then pass
through a dry filter and distil. Dis-
solve the residue in 2-3 cc. of water
and pass the solution, if necessary,
through a moist filter. Add am-
monium hydroxide solution until
alkaline and then saturated calcium
sulphate solution. If there is a pre-
cipitate, acidify with acetic acid and
9 FIG. 17. Calcium Oxalate
let solution and precipitate stand Crystals,
over night in a covered beaker. If
there is still a crystalline precipitate, it can be only calcium
oxalate. A microscopic examination of this precipitate is
advisable. Calcium oxalate forms characteristic octahedrons
having the so-called envelope-shape (Fig. 17). When thor-
oughly washed, calcium oxalate may be converted by ignition
into calcium oxide which may be weighed.
CaO :H 2 C 2 O 4 .2H 2 O = Weight of CaO :x
(56) (126) found
192 DETECTION OF POISONS
Calculation. Since the quotient 56 : 126 = 0.444, multiply the weight of
calcium oxide found by 0.444 to get the corresponding amount of crystallized
oxalic acid.
FREE ALKALIES
Potassium, Sodium and Ammonium Hydroxides
Free Alkalies. The same general principles used in detecting mineral acids
are applicable also to the alkalies. Since potassium and sodium compounds are
normal constituents of animal and vegetable organisms, and since ammonia is a
decomposition product of nitrogenous organic matter, the examination must
always show that the alkalies are in the free state, for they alone and their car-
bonic acid salts decompose and corrode animal tissues and not their neutral salts.
Poisonings due to caustic alkalies resemble those caused by corrosive acids.
If taken internally, their corrosive action gives rise to pain in the mouth, throat,
oesophagus, stomach and abdomen. Mineral acid corrosions are dry and brittle,
whereas those from caustic alkalies are soft and greasy. The alkali albuminates
formed become gelatinous, swell and may partly dissolve in presence of much
water. The destructive action of the caustic alkalies extends deep and affects
the parts around the corroded places. In caustic alkaline solutions gelatinous
tissues, horny substances, hair and skin swell considerably and finally dissolve
The stomach in alkali poisoning is softened in places, corroded and decidedly
bright red in color.
Detection of Alkalies
Ammonia
Free ammonia is usually recognized by its odor. A piece of
moist red litmus paper, held over the material, becomes blue.
A paper moistened with mercurous nitrate solution is blackened.
Distillation. If the material is strongly alkaline, extract
several times with absolute alcohol. Use a flask with a glass
stopper and distil the combined extracts. Collect the distillate
in a little dilute hydrochloric acid and evaporate the solution
to dryness in a porcelain dish upon the water-bath. Dissolve
the residue in water and test the solution for ammonia, using
Nessler's reagent and chloroplatinic acid.
Fixed Alkalies
The residue from the above distillation may contain potas-
sium and sodium hydroxides. If the residue is strongly alka-
line, first add a few drops of phenolphthalein and then excess of
barium chloride solution. The red color and the alkaline reac-
POISONS NOT IN THE THREE MAIN GROUPS 193
tion, if due to carbonates, disappear, because two neutral salts
are formed :
K 2 CO 3 + BaCl 2 = BaCO 3 + 2KC1.
But if alkaline hydroxides are present, the alkaline reaction
and red color remain, for soluble barium hydroxide is formed:
2 KOH + BaCl 2 = Ba(OH) 2 + aKCL
And the solution of this compound reddens phenolphthalein.
To distinguish potassium from sodium hydroxide, neutralize
the residue from distillation with dilute hydrochloric acid and
test for potassium and sodium as follows:
1. Add solution of chloroplatinic acid (H 2 PtCl 6 ) which
causes the precipitation of potassium in the form of the double
chloride of potassium and platinum (potassium chloroplatinate,
K 2 PtCl 6 ).
2. Add de Konink's reagent 1 which is a solution of sodium
cobaltic nitrite, 6NaNO 2 .Co 2 (NO 2 )6. This reagent produces a
yellow precipitate of potassium cobaltic nitrite, 6KNO 2 .
Co 2 (NO 2 ) 6 + xH 2 O, in a solution containing a potassium salt.
To hasten the reaction, add a few drops of acetic acid.
3. Test for sodium in a neutral solution by adding a few drops
of freshly prepared acid potassium pyro-antimonate solution,
K 2 H 2 Sb 2 O 7 . At first the solution is turbid but, if stirred, de-
posits a white crystalline precipitate of sodium pyro-antimon-
ate, Na 2 H 2 Sb 2 O 7 .
Vitali's procedure in testing for caustic alkalies consists in
shaking the alcoholic extract of the material, prepared as far as
possible with exclusion of air (see above), with freshly precipi-
tated and well-washed mercurous chloride. Free alkali black-
ens this compound. The solubility of mercurous oxide (Hg 2 O),
the black compound formed, in dilute nitric acid distinguishes
it from mercuric sulphide.
Quantitative Estimation of Hydroxides and Carbonates of
Alkalies. To determine both the free caustic alkali and that
1 Prepare sodium cobaltic nitrite by dissolving 10 grams of pure sodium nitrite
and 4 grams of cobaltous nitrate separately in sufficient water. Mix the solutions,
add 2 cc. of acetic acid and dilute to 100 cc. with water.
194 DETECTION OF POISONS
converted into carbonate, first determine total alkalinity by
titrating a portion of the distillation residue with normal or
o.i n-hydrochloric acid, using methyl orange as indicator.
Then precipitate carbonate in a second portion of the distilla-
tion residue with barium chloride solution and determine free
caustic alkali in the nitrate. If the examination shows only
alkaline carbonate, this dpes not exclude the possibility of caus-
tic alkali having been originally present.
POTASSIUM CHLORATE
Toxic Action. Large doses (4-10 grams) of potassium chlorate, KCIOs, are
decidedly toxic. During the first stage of intoxication, alteration in the shape of
the red corpuscles and conversion of oxyhaemoglobin in the intact corpuscles into
brown methaemoglobin take place. Then the red blood- corpuscles, at least in a
case of severe poisoning, change their form, becoming shriveled and undergoing
decomposition. Toxicologists (see R. Kobert, Intoxikationen) ascribe change of
blood pigment and red blood- corpuscles to specific salt action possessed in high
degree by potassium chlorate. This explanation also accounts for salt diuresis, 1
appearing at the beginning of potassium chlorate poisoning, whereby the blood is
much thickened. But most notable is the high alkalinity of the urine, resulting
in decreased alkalinity of the blood plasma. In severe chlorate poisoning so
much oxyhsemoglobin is changed to methaemoglobin that the amount of oxygen
in the blood may drop to i per cent. As a result human beings or animals thus
poisoned may become asphyxiated from lack of oxygen. Potassium chlorate
through the action of potassium weakens the heart. In chlorate poisoning the
blood has a characteristic chocolate-brown color (see above) .
Potassium chlorate taken by the mouth is quite rapidly eliminated by the
kidneys. After administration of o.i gram of potassium chlorate, chloric acid
appears in the urine in an hour. Most of the potassium chlorate passes into the
urine unchanged, only a little of the salt being reduced to potassium chloride.
During chlorate poisoning, the urine is usually very dark, even black, and may
contain haemoglobin and methaemoglobin. It is frequently opaque and strongly
alkaline. Upon long standing a dark brown sediment gradually deposits. The
urine also contains considerable albumin.
In suspected chlorate poisoning, the urine should if possible receive a thorough
chemical and microscopical examination. An anuria lasting several days may
precede death and render an examination of the urine quite impossible.
Detection of Chloric Acid
To isolate potassium chlorate from organic material, use a
dialyzer which should be as flat as possible, because the thinner
the layer in the inner container and the larger the volume of
1 Diuresis = increased secretion of urine.
POISONS NOT IN THE THREE MAIN GROUPS 195
water in the outer vessel, the more rapid the diffusion. Place
the material to be examined, as parts of organs and stomach
or intestinal contents, in the inner container of a flat dialyzer
and pure water in the outer vessel. Allow dialysis to take
place 5-6 hours without changing the water in the outer vessel.
Then evaporate the dialysate (contents of the outer vessel) to
dryness in a porcelain dish upon the water-bath. Dissolve the
residue in a little water and examine the filtered solution for
chloric acid as follows :
1. Indigo Test. Add dilute sulphuric acid and a few drops
of indigo solution, until there is a distinct blue color. Then
introduce sulphurous acid drop by drop. If chloric acid is
present, the blue color changes to yellow or greenish yellow.
This is a delicate test for chloric acid, given even by o.oi gram
of potassium chlorate.
2. Silver Nitrate Test. Add silver nitrate solution in excess.
If there is a precipitate (AgCl), filter and add a few drops of
sulphurous acid to the clear filtrate. A chlorate will cause the
precipitation of more silver chloride. Silver chloride differs
from silver sulphite in being insoluble in hot dilute nitric acid.
Sulphurous acid reduces silver chlorate to chloride:
AgClOs + 3 H 2 S0 3 = AgCl + 3 H 2 S0 4 .
3. Free Chlorine Test. A solution containing a chlorate,
heated with concentrated hydrochloric acid, gives free chlorine.
The gas passed into potassium iodide solution liberates iodine.
Shake the solution with chloroform which dissolves iodine with
a violet color. This test indicates chloric acid only in the ab-
sence of substances like chromic acid and dichromates which
also give chlorine with hydrochloric acid.
If the material is a - powder, dissolve in water and filter if
necessary. A direct test for chloric acid is usually possible with
such a solution.
Quantitative Estimation of Chloric Acid
To estimate potassium chlorate quantitatively in urine, dialy-
sates and other liquids, reduce with zinc dust, or employ
Scholtz's method.
196 DETECTION OF POISONS
1. Zinc Dust Method. Divide the solution into two equal
parts. Determine chloride gravimetrically in one portion by
precipitating and weighing AgCl, or volumetrically by titrating
according to Volhard's method.
Determine chloride and chlorate together in the second
portion. Add 5-10 grams of zinc dust and a little dilute sul-
phuric or acetic acid, and heat the mixture 0.5-1 hour upon a
boiling water-bath. Filter and wash the residue with boiling
water. Acidify the nitrate with nitric acid and precipitate
chloride with silver nitrate. More chlorine appears in the sec-
ond than in the first determination. Calculate the percentage
of potassium chlorate from the difference between the two
chlorine determinations. One molecule of KClOs upon re-
duction yields i molecule of KC1 and therefore i atom of
chlorine.
Zinc dust in presence of sulphuric or acetic acid reduces potassium chlorate to
chloride:
() KC10 3 + 3Zn = KC1 + 3 ZnO,
(0) ZnO + 2 CH S .COOH = H 2 O + Zn(CH 3 .COO) 2 .
2. Method of M. Scholtz. 1 This method makes use of the
reducing action of nitrous acid upon chloric acid :
HC10 3 + 3HN0 2 = HC1 + 3HNO 3 .
Add to the solution 10 cc. of nitric acid (sp. gr. 1.2 = 32 per
cent.) and 10 cc. of 10 per cent, sodium nitrite solution. Let
the mixture stand for 15 minutes at room temperature. Then
add 30-50 cc. of o.i n-silver nitrate solution and 5 cc. of satu-
rated iron alum solution, (I^N^SO^FesCSOOs^HsO. Ti-
trate excess of silver with o.i n-ammonium sulphocyanate
solution. 1000 cc. of o.i n-AgNO 3 = o.i KC1O 3 gram =
12.245 grams of KC1O 3 .
The slight excess of nitrous acid has no effect upon the deli-
cacy of the reaction. Liquids like dialysates of stomach con-
tents and organs always contain chloride. In that case first
1 Archiv der Pharmazie 243, 353 (1905).
POISONS NOT IN THE THREE MAIN GROUPS 197
determine the amount of chloride in another portion by Vol-
hard's method.
H. Hildebrandt 1 has adapted Scholtz's method to the ex-
amination of urine. First completely precipitate chloride in a
measured volume of urine with silver nitrate in presence of
nitric acid. Add sodium nitrite solution to the clear, chloride-
free filtrate, as well as more silver nitrate solution, until there
is no longer a precipitate. Determine as usual the weight of
silver chloride obtained.
In the case of urine a larger quantity of nitrous acid is decomposed by the urea :
CO(NH,), + 2 HN0 2 = C0 2 + 2N 2 + 2H 2 O.
Consequently do not use too little sodium nitrite.
Behavior of Potassium Chlorate in Putrefaction
C. Bischoff states that potassium chlorate, mixed with moist,
organic substances, especially blood, is very soon reduced to
chloride! Bischoff describes several cases, in which poisoning
by potassium chlorate had undoubtedly occurred, and yet
chloric acid could not be detected chemically in parts of the
cadaver.
In an experiment, too grams of blood, 0.5 gram of potassium
chlorate and 100 grams of water were allowed to stand for 5
days at room temperatures. Not a trace of chloric acid could
be detected in the dialysate. Bischoff concludes from this
experiment that potassium chlorate, mixed with moist organic
substances, especially with blood, is soon reduced. Conse-
quently, chloric acid may not be detected, even in cases of
rapidly fatal poisoning by potassium chlorate.
Detection of Chlorate in Meat
The German law of June 3, 1900, relating to the inspection of beef-cattle and
meat, forbids the use of chlorates in preserving meat, sausage and fat. The
official directions prescribed for the chemical examination of meat and fats are as
follows:
Let 30 grams of finely divided meat stand i hour in the cold with 100 cc. of
water and then heat to boiling. Filter when cold and add silver nitrate solution
1 Vierteljahrsschrift fur gerichtliche Medizin 32, 8 1 (1906).
198 DETECTION OF POISONS
in excess to the filtrate. Add 2 cc. of 10 per cent, sodium sulphite solution and
2 cc. df concentrated nitric acid to 50 cc. of the clear filtrate from the silver pre-
cipitate and then heat to boiling. If there is a precipitate, insoluble in more hot
water and consisting of silver chloride, chlorate is present.
SANTONIN, SULPHONAL AND TRIONAL
These substances do not find a place in the Stas-Otto process
on account of their behavior toward cold tartaric acid solution
and ether. Use the following method for their detection.
Extract the material, neutral or faintly acid with tartaric
acid, under a reflux condenser with boiling absolute alcohol.
Filter hot and evaporate the filtrate to dryness upon the water-
bath. Dissolve the residue in hot water. If the solution is
colored, digest for some time upon the water-bath with bone-
black and stir frequently. Filter the hot solution. All of the
above substances, if present in considerable quantity, crystallize
in part as the solution cools. -Extract the filtrate and any
crystals thoroughly with chloroform several times. Pass the
chloroform extract through a dry filter. The residue from
chloroform may contain santonin, sulphonal and trional, as
well as acetanilide and phenacetine.
The chloroform residue may also contain those substances
extracted in the Stas-Otto process from the acid solution by
ether. Chloroform completely extracts substances like anti-
pyrine, caffeine, acetanilide, phenacetine and salicylic acid.
As a rule they are purer from this solvent than from ether.
The chloroform residue may also contain the weak base narco-
tine.
SANTONIN
Santonin, CuH 18 Oj, crystallizes in colorless, inodorous, shining leaflets which
are bitter and melt at 170. Santonin dissolves in 5000 parts of cold and 250
parts of boiling water; in 44 parts of ethyl alcohol; and in 4 parts of chloroform.
All these solutions are neutral. It is slightly soluble in ether (1:150). Light
turns these crystals yellow. Upon evaporation, an alcoholic solution of the
yellow modification deposits white santonin.
Constitution. Santonin is the internal anhydride (lactone) of santonic acid,
CuH 2 oO 4 . Caustic alkalies, as well as calcium and barium hydroxides, dissolve
santonin forming salts of this acid. In this case, as with all lactones, the lactone
ring is broken as follows :
POISONS NOT IN THE THREE MAIN GROUPS 199
CH 3 CH 3
c c 2 c c 2
H 2 C C CH O\ H. HzC c CH OH
>CO + OK = | | |
OC C CH CH/ OC C CH CH COOK
\/ V I \/ \/ I
C C CH 3 C C CH 3
H 2 | H 2
CH 3 CH 3
Santonin Potassium santonate
A solution of a santonate, acidified with hydrochloric acid, first gives free
santonic acid. To isolate this compound from the mixture, extract at once with
ether. Otherwise, the acid loses i molecule of water upon standing and passes
into its internal anhydride, santonin.
Santonin is also a ketone. As such it forms a hydrazone, CisHi 8 O 2 = N-
NH.CsHs, with phenylhydrazine and an oxime, CuHisOz = NOH, with hy-
droxylamine.
According to the structural formula above, santonin is a derivative of hexa-
hydro-dimethyl-naphthalene. Fused with potassium hydroxide, santonic
acid gives hydrogen, propionic acid and a naphthalene derivative, namely,
dimethyl-/?-naphthol.
Behavior in the Organism. Santonin seems to be incompletely absorbed in the
body. M. Jaffe 1 has administered quite large quantities of santonin to dogs and
rabbits. He obtained a new substance, called a-oxysantonin (CnHuO<), from
the urine of the dog, amounting to 5 or 6 per cent, of the santonin administered.
He extracted with chloroform considerable quantities of unaltered santonin from
the faeces of the dog. Rabbits can usually tolerate being fed with santonin for
weeks, and a-oxysantonin is formed only in very small quantity. In the ether
extract of the rabbit's urine, Jaffe found a second santonin derivative, /3-oxy-
santonin, isomeric with a-oxysantonin, with considerable unaltered santonin. In
these experiments only about half the santonin administered was absorbed by the
rabbit.
After administration of santonin, a red pigment called santonin red appears in
human urine. Even after medicinal doses santonin urine is red, or becomes at
least scarlet-red to purple on addition of potassium or sodium hydroxide solution.
Urine containing santonin also becomes carmine-red on addition of calcium hy-
droxide solution.
Detection of Santonin
Ether, benzene, or better chloroform, extract santonin only
from acid solutions. The organic solvent fails to remove this
compound from an alkaline solution, as it is then in the form
of a santonate. Santonin is not an alkaloid and forms no pre-
x Zeitschrift fur physiologische Chemie 22, 537 (1896-1897).
200 DETECTION OF POISONS
cipitates with the general alkaloidal reagents, but it gives
several more or less characteristic color reactions.
1. Alcoholic Potassium Hydroxide Test. Pure santonin,
heated with an alcoholic solution of potassium hydroxide, gives
a fine carmine-red color, which gradually changes to reddish
yellow and finally fades entirely. In this test yellow santonin
dissolves at once with a yellowish red color.
2. Sulphuric Acid-Ferric Chloride Test. Heat santonin with
concentrated sulphuric acid and add a drop of ferric chloride
solution. The mixture becomes violet. Use about i cc. of
sulphuric acid to o.oi gram of santonin.
3. Furfural-Sulphuric Acid Test. Mix 2-3 drops of alcoholic
santonin solution with 1-2 drops of 2 per cent, alcoholic fur-
fural solution and 2 cc. of pure concentrated sulphuric acid.
Warm this mixture in a small porcelain dish upon the water-
bath. A purple-red color appears and changes with continued
heating to crimson-red, blue-violet and finally to dark blue
(Thater 1 ).
Only a few alkaloids and glucosides give distinct color reactions with furfural
and sulphuric acid. Substances behaving similarly are veratrine, picrotoxin
(violet) and piperine (green to blue-green, finally indigo-blue). The colors given
by a- and /3-naphthol with furfural and sulphuric acid are also characteristic.
SULPHONAL
Sulphonal, C?Hi 50482, crystallizes in colorless, inodorous and tasteless prisms,
melting at 125-126 and distilling with slight decomposition at 300. It is soluble
in 500 parts of cold and 15 parts of boiling water; in 135
C H! > parts of ether; and in 65 parts of cold and 2 parts of
CH 3 C SO 2 C 2 H 6 boilm 8 eth yl alcohol. Sulphonal is freely soluble in
chloroform. Especially characteristic of this compound
SO 2 .C 2 H 6 are the ease with which it crystallizes and its great
stability in presence of chemical reagents. The halogens, halogen acids, alkaline
hydroxides and carbonates, concentrated sulphuric and nitric acids are without
action in the cold.
Preparation. The condensation of ethyl mercaptan (2
molecules) with acetone (i molecule) by means of dry hydrogen
chloride gas, or concentrated sulphuric acid, results in the
formation of the ethyl-mercaptole of acetone. The latter com-
1 Archiv der Pharmazie 235, 410 (1897).
POISONS NOT IN THE THREE MAIN GROUPS 201
pound, shaken with a saturated solution of potassium perman-
ganate in presence of dilute sulphuric acid, undergoes oxidation
with formation of sulphonal: 1
H 3 C X HSC 2 H 5 H 3 C\ /SC 2 H 5 + 2 H 3 C\ /SO 2 C 2 H 6
>C = O + =H 2 O+ C > C
H,(X HSC 2 H 6 H 3 C/ \SC 2 H 5 + O 2 H 3 C/ \SO 2 C 2 H 5
Acetone Ethyl Ethyl-mercaptole Sulphonal
mercaptan of acetone
Detection of Sulphonal
Ether, or better chloroform, extracts sulphonal from acid,
neutral and alkaline solutions. Test the residue left upon
evaporating these solutions as follows :
1. Melting-point Test Determine the melting point (125-
126) of the perfectly pure substance. Crystallization from
boiling water with the use of a little bone-black easily gives a
pure product. A mixture of these crystals with pure sul-
phonal should also melt at 125-126.
2. Reduction Test. Sulphonal heated in a test-tube with
powdered wood charcoal gives the characteristic odor of ethyl
mercaptan.
3. Detection of Sulphur. (a) With Sodium. Fusion of
sulphonal in a dry test-tube with a little metallic sodium pro-
duces sodium sulphide. Dissolve cautiously (unaltered metallic
sodium!) the cold melt in a little water, filter and test the
filtrate with sodium nitroprusside solution for sulphide (see
page 23).
(6) With Potassium Cyanide. Fuse i part of sulphonal
and about 2 parts of pure potassium cyanide in a dry test-
tube. Note the penetrating odor of ethyl mercaptan (C 2 H 5 .SH).
Potassium sulphocyanate is also a product of the reaction. An
aqueous solution of the melt, acidified with dilute hydrochloric
acid, becomes deep red with 1-2 drops of ferric chloride solution.
(c] With Powdered Iron. Sulphonal heated with pure pow-
dered iron free from sulphur gives a garlic-like odor. Add
1 Sulphur in the sulphone group = SO 2 is most likely sexivalent, corresponding
to the atomic grouping I, and not quadrivalent, as in II:
VI /& IV /O
i. = sf ; n. = s< |
X) X O
202 DETECTION OF POISONS
hydrochloric acid to the residue. Hydrogen sulphide evolved
blackens lead acetate paper.
Detection of Sulphonal in Urine
Sulphonal is cumulative in its action. Therefore continuous administration
for a long time of large doses may result in the collection of a considerable
quantity of this compound in the organism. Most of the sulphonal taken ap-
pears in the urine as ethyl-sulphonic acid, C2H5-SO2OH. 1 The formation of
this acid causes an increase of ammonia in the urine during sulphonal intoxication,
as does administration of mineral acids.
Sulphonal occurs in urine in detectable quantity only following considerable
doses, especially when they have been taken without interruption. Such urine
is often dark red to garnet-brown from haematoporphyrin. But this decom-
position product of blood pigment appears in urine only succeeding severe
poisoning by sulphonal, and even then its occurrence is rare.
To isolate sulphonal from urine, evaporate 1000 cc. to one-tenth its volume,
and extract several times with large quantities of ether. Pass the ether extracts,
after they have settled in a dry flask for several hours, through a dry filter and
distil. Evaporate the residue with 20-30 cc. of 10 per cent, sodium hydroxide
solution to dryness upon the water-bath. This will remove coloring matter,
extracted from urine by ether, but will not affect the sulphonal. Extract sul-
phonal from the alkaline residue with ether. Evaporate the solvent, and sul-
phonal will remain pure and almost colorless. Determine the melting-point of
this residue, and make the other tests for sulphonal.
Detection of Haematoporphyrin in Urine
Coloring matters have been observed in red, brownish red to cherry-red
urines, which quite probably are identical with haematoporphyrin. The
spectroscopic examination of such urine is made in the following manner. Add
sodium hydroxide solution, drop by drop, to about 500 cc. of urine, until the
reaction is strongly alkaline, and then add a little barium chloride solution.
Filter after a while, and wash the precipitate well. Extract the precipitate
upon the filter with hot alcohol, containing a few drops of dilute sulphuric acid.
A spectroscopic examination of this filtrate can be made directly with a Brown-
ing pocket spectroscope. Acid haematoporphyrin solutions are violet; when
more concentrated, they have a cherry-red color, and show the characteristic
spectrum with two absorption-bands (see page 306). If the acid, alcoholic
solution is saturated with a few drops of ammonium or sodium hydroxide
solution, the spectrum of alkaline haematoporphyrin solution with its four ab-
sorption-bands appears. Traces of hsematoporphyrin very frequently appear
1 The structural formula of ethyl-sulphonic acid is: C 2 H 6 .S^OH.
V>
It should not be confused with ethyl-sulphuric acid: C 2 H 6 -O-S^OH.
V)
POISONS NOT IN THE THREE MAIN GROUPS 203
in normal urine. It has been observed more abundantly, at times, in urine
during chronic sulphonal poisoning.
TRIONAL
Trional crystallizes in colorless, shining leaflets melting at 76. It is soluble in
320 parts of cold, but more easily soluble in hot water. It is also soluble in ethyl
alcohol, ether and chloroform. The aqueous solution is
\ c / neutral and bitter. In the latter respect it differs from
C 2 jj 6 / \S0 2 C 2 Hj sulphonal which is tasteless. Trional gives the sulphonal
reactions. Trional is completely decomposed in the
organism and the danger of cumulative action is much less than in the case of
sulphonal. Moreover, haematoporphyrin has almost never been observed, even
following considerable doses of trional and after uninterrupted use for weeks.
Active Organic Substances 1 Rarely Occurring in lexicological Analysis
CANTHARIDIN
Cantharidin, CioHi 2 O4, is the active vesicating principle of Spanish fly
(Lytta vesicatoria) and is present to the extent of 0.8-1 per cent. Cantharidin
H forms colorless, shining, neutral, rhombic leaflets,
C CHs melting at 218 and subliming at higher temperature
in white needles. It is almost insoluble even in boiling
H 2 C
I
H 2 C !
C = O
water. Acids, as tartaric acid, increase its solubility
yO in water, though Cantharidin is not a base. It dis-
I c = O solves with difficulty in cold ethyl alcohol (0.03 : 100 at
\)X \ 18) and in ether (0.011:100). Chloroform (1.52:100),
C CHs acetone and acetic ether are its best solvents. It is
as good as insoluble in petroleum benzine.
Constitution. According to Gadamer 2 Cantharidin has the structural formula
shown above. Treated with potassium or sodium hydroxide, it loses its anhy-
dride character and passes into solution as the alkali salt of dibasic cantharidic
acid, CioHi 4 O 5 :
H H
C CHs C CHs
' /|\ /
C C = O H 2 C C COOK
H 2 C
1
>o
KiOH =
O
H 2 C
C C =
6
j H
H 2 C
C COOK
\
/
' \
\
/
' \
C
CH 3
OK
CHs
H
H
Cantharidin
Potassium cantharidat
H 2 0.
Potassium cantharidate, CioHi 2 O 5 K 2 .2H 2 O, recently recommended for
phthisis, and sodium cantharidate, Ci Hi 2 O 6 Na 2 .2H 2 O, are well crystallized
1 The toxic substances considered in this place have been arranged in alpha-
betical order.
2 Chemical Abstracts 12, 806 (1918).
204
DETECTION OF POISONS
salts. Mineral acid first sets cantharidic acid free from these salts. The latter
soon loses a molecule of water, passing into its internal anhydride, cantharidin.
H 2 C
H
C
/\
/ CH 3
^C COOiH
H 2 C
C COjOH
\/
C
II
H
C
/ \
CH 3
H,C X
\ /
C C =
<
>
H 2 C
C C =
MX
\
C
CH 3
+ H 2
Cantharidic acid
Cantharidin
Cantharidin, heated with hydriodic acid at 100, or treated at room tem-
perature with chlorosulphonic acid, Cl-SOj-OH, changes into the isomeric
cantharic acid, CioHi2O4, crystallizing in colorless needles melting at 275
and having the following structure:
HC
H 2 C C COOH
V
H,
Cantharic acid
This acid is not a vesicant. Heated for 3 hours at 135 in sealed tube with
acetyl chloride, cantharic acid yields another isomer of cantharidin which Ga-
damer 1 has shown to be acetyl-hydrato-cantharic anhydride having the following
structure:
H
C CH 3
/\ /
HC C C = O
H 2 C C C = O
\/ \
C CH 3
/\
H CO CH 3
Acetyl-hydrato-cantharic anhydride
The latter crystallizes from alcohol in large colorless leaflets melting at 76.
There is a close relationship between o-xylene and cantharidin, for the latter,
heated at 400 with calcium hydroxide, gives a dihydro-o-xylene, C 8 Hi 2 , called
cantharene, and also o-xylene, C 6 H 4 (CH 3 ) 2 , and xylic acid. Finally, cantharidin,
heated with an excess of phosphorus pentasulphide and distilled, gives pure
o-xylene. (J. Piccard.) 2
1 Chemical Abstracts 12, 806 (1918).
2 Berichte der Deutschen chemischen Gesellschaft 12, 577 (1879).
POISONS NOT IN THE THREE MAIN GROUPS 205
Detection of Cantharidin
Evaporate a liquid, or material containing much moisture
(organs, stomach or intestinal contents, etc.), to dryness upon
the water-bath. Dragendorff directs repeated extraction of the
finely divided material with alcohol containing sulphuric acid.
Filter the extracts, add one-sixth their volume -of water and
distil the alcohol. Extract the residue 2-3 times with chloro-
form and shake the chloroform extracts with water to remove
adherent acid. Finally separate the chloroform from water,
distil and examine the residue for cantharidin. Since this com-
pound gives no characteristic chemical reactions, employ the
physiological test for identification. Dissolve the chloroform
residue, unless fatty substances are present, in a few drops of
hot almond oil. Bind a cloth, saturated with this solution,
upon the upper arm or breast by means of adhesive plaster.
Cantharidin reddens the skin and sometimes raises blisters.
Even 0.14 mg. of cantharidin causes blistering. Salts of can-
tharidic acid also have a vesicating action.
To detect cantharidin in blood, brain, liver and other material
rich in protein, E. Schmidt boils with dilute potassium hydrox-
ide solution (i gram of KOH in 15 cc. of water), until the mass
is homogeneous, acidifies with dilute sulphuric acid and extracts
thoroughly with hot alcohol. The procedure in other respects
is as described above.
Cantharidin is said to resist putrefaction.
CYTISINE
Cytisine, CiiHuN 2 O, occurs in the ripe seeds of Golden chain (Cytisus Labur-
num) which contain about 1.5 per cent. Cytisine and the alkaloid originally
called ulexine, isolated from the seeds of Ulex europaeus, are identical (A.
Partheil).
Preparation. Extract powdered ripe laburnum seeds with 60 per cent, alcohol
containing acetic acid. Distil the alcohol from the extracts, pour the residue
through a moist filter and precipitate extractive and tannin substances with
lead acetate solution. Filter, add potassium hydroxide solution to the clear
nitrate and extract cytisine with chloroform. Distil the chloroform which
usually deposits cytisine as a radiating crystalline mass. If purification is neces-
sary, recrystallize the residue from absolute alcohol or boiling ligroin. Sub-
limation in a partial vacuum also purifies crude cytisine.
206 DETECTION OF POISONS
Cytisine crystallizes in large, colorless, tasteless prisms, melting at 152-153
and subliming at a higher temperature, if carefully heated. It dissolves freely
in water, alcohol, chloroform and acetic ether; less easily in commercial ether,
benzene and acetone; and is insoluble in petroleum ether and absolute ether.
Cytisine is a strong secondary base and very toxic. Although capable of com-
bining with i or 2 molecules of hydrochloric acid, this compound behaves in
other respects like a monacid base. Only the salts containing one equivalent
of acid crystallize well. Nitrous acid converts this secondary base into nitroso-
cytisine, CnHisON-NO, which crystallizes in needles. Nitrous fumes appear,
if cytisine is warmed upon the water-bath with twice the amount of concentrated
nitric acid, and the solution at once becomes reddish yellow to brown. This
solution poured into water gives a precipitate of nitro-nitroso-cytisine, CnHi 2 ON-
(NO Z )N-NO. This compound crystallizes from water in pale yellow scales
melting at 242-244.
Toxic Action. Cytisine produces convulsions, its action in this respect being
very similar to that of strychnine. But unlike the latter alkaloid it also irri-
tates the gastro-intestinal mucosa even causing bloody inflammation. Cytisine
also differs from strychnine in stimulating the vomiting center. Consequently,
after doses of cytisine or laburnum preparations, human beings and animals
capable of emesis thus rid the organism of a large part of the poison. Like
strychnine, cytisine stimulates the respiratory and vaso-motor centers. Finally
as in strychnine intoxication death results from paralysis of these two centers.
A part of the cytisine leaves the organism unchanged and appears in the urine
(R. Robert).
Detection of Cytisine
Prepare an aqueous tartaric acid solution of stomach con-
tents, vomitus or parts of organs, following the general pro-
cedure for alkaloids. To remove final traces of fatty acids and
fat, shake this solution well with ether. Withdraw the aqueous
solution, make alkaline with sodium hydroxide solution and
extract thoroughly with chloroform or isobutyl alcohol. Evap-
orate the chloroform or isobutyl alcohol extracts and test the
residue as follows for cytisine:
1. Van der Moer's 1 Test Ferric chloride solution colors
cytisine and its salts blood-red. Dilution with water, or acidi-
fication, discharges this color. Hydrogen peroxide also pro-
duces the same result. The solution containing hydrogen
peroxide, warmed upon the water-bath, becomes intensely
blue.
2. A. Ramverda's 2 Test. A little nitrobenzene, containing
1 Berichte der Deutschen pharmazeutischen Gesellschaft 5, 267 (1895).
2 Chemisches Zentral-Blatt, 1900, II, 268.
POISONS NOT IN THE THREE MAIN GROUPS 207
dinitro-thiophene, poured upon cytisine gives a fairly stable,
brilliant red- violet color.
A similar color given by coniine is very unstable.
3. Nitro-Nitroso-Cytisine Test. Nitro-nitroso-cytisine (see
above), formed by concentrated nitric acid, serves to detect
small quantities of this alkaloid. Nitro-nitroso-cytisine dis-
solves with difficulty in 94 per cent, alcohol and crystallizes
from this solvent in microscopic prisms. Flat, tabular crystals
form from 50 per cent, alcohol which is a better solvent. The
solubility of nitro-nitroso-cytisine in concentrated hydrochloric
acid indicates basic properties, but they are feeble, for dilution
with water precipitates this compound unchanged.
THE DIGITALIS GLUCOSIDES
The digitalis plant (Digitalis purpurea L.) contains in all its
parts, but especially in the leaves and seeds, medicinally useful
substances belonging to the glucoside group. Thus far three
digitalis glucosides have been isolated as well characterized,
crystalline compounds of homogeneous composition. These are
digitalin in a narrower sense (= Digitalinum verum crystal-
lisatum Kiliani) C 3 5H 56 Oi4; digitoxin, C 34 H54Oii; and digitonin,
C 55 H94O 2 8 or C 5 4H9 2 O 2 8. A fourth glucoside called digitalein
seems not to have been obtained wholly pure as yet.
Digitonin, CssH^C^s or C54H92O28, 1 occurs almost exclusively
in digitalis seeds, the leaves containing at most only traces.
Digitonin, classified at present with the saponins (see page 220),
crystallizes from alcohol in fine needles soluble in 50 parts of
50 per cent, alcohol. Even very dilute hydrochloric acid
hydrolyzes digitonin into digitogenin, dextrose and galactose: 2
2 H 2 O = CsiHsoOe + 2C 6 H 12 8 + 2C 6 H 12 O(?).
Digitonin Digitogenin Dextrose Galactose
Digitonin crystallizes from alcohol in fine needles which soften
at 235 and become yellow. Digitonin is not a cardiac poison.
Pure digitonin and concentrated sulphuric acid, upon addition
1 The results obtained by A. Windhaus (Berichte der Deutschen chemischen
Gesellschaft 42, 238 (1909) favor the formula C^^tOzs for digitonin.
2 H. Kiliani, Berichte der Deutschen chemischen Gesellschaft 24, 340 (1891).
208 DETECTION OF POISONS
of a little bromine water, give a color which becomes intensely
red.
Digitoxin, C 3 4H 5 4Oii, occurs almost exclusively in digitalis
leaves. This very active and highly toxic compound is almost
wholly insoluble in water and ether but soluble in alcohol and
chloroform. Consequently ether precipitates it from chloro-
form solution. Digitoxin crystallizes from 85 per cent, alcohol
in leaflets melting at 145. Alcoholic hydrochloric acid hy-
drolyzes it forming digitoxigenin and digitoxose :
C 3 4H 5 4p u + H 2 O = C 22 H 32 04 + 2C 6 H 12 4 .
Digitoxin Digitoxigenin Digitoxose
Digitoxin dissolves in concentrated sulphuric acid with a
brownish or greenish brown color unchanged by bromine.
Kiliani's Digitoxin Test. 1 Dissolve a trace of digitoxin in
3-4 cc. of glacial acetic acid containing iron (100 cc. of glacial
acetic acid and i cc. of 5 per cent, ferric sulphate solution).
Cautiously add sulphuric acid containing iron (100 cc. of sul-
phuric acid and i cc. of 5 per cent, ferric sulphate solution) in
about the same quantity as an under layer. A dark zone ap-
pears where the two solutions meet, above which after a few
minutes a blue band is visible. After some time the entire
acetic acid layer becomes deep indigo-blue.
Digitalin, CssHseO^, according to Kiliani occurs only in
digitalis seeds. It is soluble in water (i : 1000) and very active.
Boiling with very dilute hydrochloric acid hydrolyzes its alco-
holic solution into digitaligenin and two sugars, namely, dex-
trose and digitalose : 2
CssHseOn + H 2 O = C 22 H 30 q 3 + C 6 Hi 2 O 6 + aCrHuOs
Digitalin Digitaligenin Dextrose Digitalose
Test for digitalin as follows :
i. Concentrated sulphuric acid colors pure digitalin orange-
yellow. This solution soon becomes blood-red, changing upon
addition of a little bromine water to cherry and blue-red. A
drop of nitric acid or ferric chloride solution will do as well as
bromine water. This test after 1-2 hours is surer and more
1 Archiv der Pharmazie 234, 273-277 (1896).
2 H. Kiliani, Berichte der Deutschen chemischen Gesellschaft 31, 2454 (1898).
POISONS NOT IN THE THREE MAIN GROUPS 209
permanent, if a trace of digitalin is dissolved direct in concen-
trated sulphuric acid and nothing else is added.
2. Concentrated hydrochloric acid dissolves digitalin with a
golden yellow color, changing with heat to garnet or violet-red.
At present nothing definite is known regarding the fate of digitalis glucosides
in the human organism, or the products into which they are changed or the
forms in which they are eliminated. In the case of human beings elimination
of the three active substances has never been observed. Moreover, R. Robert
has not been able to detect anything active in the urine of animals except in
isolated cases. Thus far it has not been possible to find any of the digitalis
compounds mentioned above in blood or animal organs. In a toxicological
analysis especial attention would have to be given to vomitus and the contents
of the gastro-intestinal tract. But there is slight chance of detecting the digitalis
bodies in such material.
ERGOT
Officinal ergot (Secale cornutum) is the sclerotium (compact mycelium or
spawn) of Claviceps purpurea collected from rye shortly before the fruiting period
and dried at gentle heat. Ergot is commonly known as an abortifacient and in-
toxications have occurred from its use. Consequently examinations for legal pur- .
poses may require its detection in powders and other mixtures. Our knowledge
of the constituents of ergot is still very defective notwithstanding several ex-
haustive investigations. Ergot alkaloids, as ergotine, ergotmine, cornutine,
picro-sclerotine, were described long ago. But, with the possible exception of
ergotinine (Tanret, C. C. Keller), the preparations were not entirely pure. Ergot
contains in addition to alkaloids other peculiar chemical substances which have
received but little attention. They have not the characteristic physiological
action of ergot but, like the pigment sclererythrin, are useful for purposes of identi-
fication. Among these substances belong sphacelic acid and sclerotic acid, ac-
cording to R. Robert a very poisonous resin having acid properties.
Alkaloids. The most recent 1 researches upon ergot mention as well character-
ized bases ergotinine, CasHagNsOs, and hydro-ergotinine, CssH^NsOe. Barger
calls the latter ergotoxine. Ergotinine crystallizes from alcohol in long needles
melting at about 229 when heated rapidly. This compound dissolves in 52
parts of boiling alcohol; in i.i parts of ether; and is readily soluble in chloroform.
Crystalline salts of this base have not yet been prepared. Hydro-ergotinine
(= hydrate of ergotinine), obtained as a crystalline phosphate from ergotinine
mother liquors by means of alcohol and phosphoric acid, is a white powder soften-
ing at 155 and melting at 162-164. Though freely soluble in alcohol, it dis-
solves but slightly in ether. As a rule the salts of hydro-ergotinine (ergotoxine)
crystallize well. 2 By preparing a cold methyl alcohol solution of hydro-ergo-
tinine and boiling this solution for several hours under a return condenser, F.
1 F. Rraft, Archiv der Pharmazie 244, 336 (1906) and G. Barger, Journal of
the Chemical Society 91, 337.
2 G. Barger and F. H. Carr, Proceedings of the Chemical Society 23, 27.
14
210 DETECTION OF POISONS
Kraft has converted this substance completely into ergotinine. On the other
hand, ergotinine in dilute acetic acid solution passes back almost entirely into
hydro-ergotinine within 10 days. As an indication of purity, a solution of hydro-
ergotinine in 2 parts of cold methyl alcohol after several days standing should not
deposit crystals (ergotinine) nor become green. Solutions of both alkaloids are
fluorescent. According to Keller the play of colors with sulphuric acid and ferric
chloride is characteristic of ergotinine (see below).
Physiological Action of the Alkaloids. Ergotinine and hydro-ergotinine ac-
cording to A. Jaquet produce convulsions and gangrene. They are not, however,
the cause of the specific uterine contraction characteristic of ergot. Keller's
coroutine according to Kraft is identical with ergotinine, according to G. Barger
and H. H. Dale 1 with ergotinine, which is impure from ergotoxine (hydro-
ergotinine). The English investigators believe that the physiological effects
observed with ergotinine are due to adhering ergotoxine. The latter is readily
formed when the difficultly soluble ergotinine is brought into solution by means of
glacial acetic acid, phosphoric acid, or a little sodium hydroxide solution. Ergo-
toxine according to Barger and Dale produces the effects typical of ergot, causing
powerful contraction of the uterus and later abortion.
Sclererythrin. This is the pigment of the outer coat of ergot. E. Schmidt
gives the following directions for its isolation. Extract freshly powdered ergot
with ether to remove fat. Then moisten the powder with water containing tar-
taric acid, dry and extract with 95 per cent, alcohol. Filter and distil the alcohol.
Extract the residue with ether. This solvent now dissolves sclererythrin which
can be precipitated by means of petroleum ether.
Sclererythrin is an amorphous red powder which can be sublimed. It is in-
soluble in water but soluble in absolute alcohol and glacial acetic acid. This
substance behaves like an acid, dissolving in caustic alkalies, ammonia, and
alkaline carbonate and bicarbonate solutions with a red or red-violet color.
Owing to presence of sclererythrin, ether, if shaken with powdered ergot mois-
tened with tartaric acid solution, becomes red. If such an ether solution of
sclererythrin is shaken with sodium hydroxide solution, the pigment dissolves in
the latter which then becomes red. Solutions of this pigment show characteristic
absorption- bands in the spectrum. Moreover the pigment gives blue-violet
precipitates with solutions of calcium hydroxide, barium hydroxide and lead
acetate. The precipitate with stannous chloride is currant-red; with copper sul-
phate a pure violet; with ferric chloride a deep green; and with chlorine or bro-
mine water a lemon-yellow.
Detection of Ergot in Flour, Bread and Powders
This examination usually consists in undertaking to detect
by chemical and physical means the red pigment sclererythrin
which is characteristic of ergot. The property possessed by
this substance of passing from ether into a solution of an alkaline
hydroxide or bicarbonate is especially valuable for purposes of
identification.
1 Bio-Chemical Journals, 240.
POISONS NOT IN THE THREE MAIN GROUPS 211
1. Detection of Sclererythrin. Shake frequently and let 10
grams or more of flour stand for a day in a closed flask with 20
cc. of ether and about 15 drops of dilute sulphuric acid (i : 5).
Then pass the ether through a dry paper, wash the residue with
a little ether and shake the filtrate thoroughly with 10-15 drops
of cold saturated sodium bicarbonate solution. If the flour
contains ergot, the aqueous layer separates with a violet color.
R. Palm extracts the flour at 3040 with 10-15 times its
volume of 40 per cent, alcohol containing a few drops of am-
monia. Express the liquid, filter and add basic lead acetate
solution to the filtrate. Press the precipitate between filter
paper and warm while still moist with a little cold saturated
borax solution. A red-violet color appears, if the flour contains
ergot.
2. Spectroscopic Examination. This test gives a positive
result, if the material (powdered ergot, flour, bread) contains
more than o.i per cent, of ergot. Examine spectroscopically
the alkaline and acid solution of the pigment. The red solu-
tion, prepared in Test i by means of ether containing sulphuric
acid, shows two absorption-bands. One lies in the green be-
tween ,E and F but nearer E and a second broader band in the
blue midway between F and G. Then render the solution
alkaline with ammonia. Three absorption-bands should appear.
The first lies between D and E, the second at E somewhat to the
right and the third to the left of F.
3. Choline. Ergot powder, warmed with dilute potassium
hydroxide solution, gives the characteristic odor of trimeth-
ylamine, (CH 3 ) 3 N, 1 due to decomposition of choline in ergot.
/CH 2 .CH 2 .OH
(CH 3 ) 3 N<
\OH
Occasionally flour that does not contain ergot may give an
odor when heated with potassium hydroxide solution.
4. Detection and Quantitative Estimation of Ergotinine (C. C.
Keller). Dry finely powdered ergot over lime, place 25 grams
1 The so-called corn smut (Ustilago Maidis), said to cause effects similar to
those of ergot, also gives the trimethylamine odor when warmed with potassium
hydroxide solution, for it contains appreciable quantities of choline.
212 DETECTION OF POISONS
in a Soxhlet tube and completely extract fat with petroleum
ether. Dry the powder at gentle heat, add 100 grams of ether
and after 10 minutes shake well with milk of magnesia (i gram
of MgO and 20 cc. of water) . Shake repeatedly during an hour
and then pass 80 grams of the ether solution (=20 grams of
ergot) through a covered folded filter into a separating funnel.
Shake the ether in succession with 25, 15, 10 and 5 cc. of 0.5
per cent, hydrochloric acid. Pour the hydrochloric acid ex-
tracts, now containing the ergot alkaloids, through a small
moistened filter. l Add ammonia until alkaline and extract the
solution twice with about half its volume of ether. Let the ether
extract settle in a dry flask, then filter into a dry weighed flask
and wash the filter with a little ether. Distil the ether and
dry flask and residue at 100 to constant weight. Good Ger-
man ergot contains 0.13-0.16 per cent, and Russian ergot 0.22-
0.25 per cent, of the alkaloid.
To detect ergot alkaloid qualitatively, proceed as follows:
(a) Dissolve a part of the residue in i cc. of concentrated
sulphuric acid and add a trace of ferric chloride solution. The
mixture is orange-red and becomes at once deep red but the
margin appears bluish to bluish green.
(b) Dissolve a part of the residue in about 4 cc. of glacial
acetic acid and add a trace of ferric chloride solution. Cau-
tiously add this mixture to concentrated sulphuric acid as an
upper layer. If ergotinine is present, a brilliant violet color
appears where the two liquids meet.
OPIUM
Detection of Meconic Acid and Meconine
Since it is comparatively easy to procure small quantities of
opium preparations, especially the tincture, poisoning from this
source is possible. Consequently, it is often desirable to
recognize the presence of opium itself. Detection of the
alkaloids narcotine and morphine, always present in opium in
1 Clarify the filtrate from these hydrochloric acid extracts, if not clear, by
agitation with a little talcum powder, previously treated with hydrochloric acid
and thoroughly washed with water. Then filter again.
POISONS NOT IN THE THREE MAIN GROUPS 213
considerable quantity, affords partial evidence of the presence
of this substance. Moreover, opium always contains two non-
basic substances, meconic acid and meconine. Detection of
these two compounds in conjunction with narcotine and
morphine definitely determines the presence of opium.
Meconic Acid, C 7 H 4 O 7 = C 5 HO 2 (OH)(COOH) 2 , is an oxy-
pyrone-dicarboxylic acid (II) and therefore a derivative of
pyrone (I) :
o o
c c
HC CH HC C OH
I. || || II.
HC CH HOOC C C COOH
Y V
Pyrone Meconic acid
Meconic acid crystallizes in plates or prisms with 3 mole-
cules of water. It is easily soluble in hot water and alcohol.
A solution of a ferric salt turns a meconic acid solution dark r,ed.
To detect meconic acid, extract a portion of the material
with alcohol containing a few drops of hydrochloric acid. Filter
and evaporate the filtrate upon the water-bath. Dissolve the ,
residue in a little water and heat the filtered solution to boil-
ing with excess of calcined magnesium oxide. The solution
contains magnesium meconate. Filter hot to remove undis-
solved magnesium oxide, evaporate the filrtate to a small
volume and acidify faintly with dilute hydrochloric acid. Add
a few drops of ferric chloride solution. A blood-red color
appears, if meconic acid is present. Wanning with hydro-
chloric acid does not discharge this red color, in which respect
it differs from the red color caused by acetic acid. This color
differs from that caused by sulphocyanic acid in not being
affected upon addition of gold chloride. But stannous chloride
reduces ferric to ferrous oxide and discharges the color. Nitrous
acid, however, at once restores it.
These tests permit the identification of meconic acid in an
extract from only 0.05 gram of opium.
Meconine, CioHi 4 . Opium contains only 0.05-0.08 per
cent, of this compound. It forms small prisms, melting at 102
214 DETECTION OF POISONS
and subliming at higher temperature without decomposition.
Meconine dissolves freely in alcohol, ether, benzene and chloro-
form, but less easily in water. Alkalies convert meconine
into easily soluble salts of meconinic acid, Ci Hi2O 5 . This
monobasic acid cannot exist free but changes to meconine when
liberated from its salts by a mineral acid. Meconine, formed
by abstracting a molecule of water from meconinic acid, is
therefore the internal anhydride (lactone) of meconinic acid:
CHr- 0;H CH 2 O
i ! i
HC C CO iOH HC C CO
HC C OCH 3 HC C OCH 3
C C
OCHa OCH 3
Meconinic acid Meconine
To detect meconine, extract the material with alcohol con-
taining sulphuric acid. Filter and evaporate the filtrate to a
syrup upon the water-bath. Dissolve the residue in water and
extract meconine from this acid solution with benzene. Evapo-
ration of the solvent frequently gives crystals of meconine. To
detect meconine, dissolve in a little concentrated sulphuric acid.
The solution is green but turns red within two days. If the
green sulphuric acid solution, or that which has turned red upon
standing, is carefully warmed, a fine emerald-green color ap-
pears, passing through blue, violet and finally back to red.
Selenious-Sulphuric Acid Reagent for Opium Alkaloids 1
Prepare the reagent used in these tests by dissolving 0.5
gram of selenious acid (H 2 Se0 3 ) in 100 grams of pure concen-
trated sulphuric acid. This reagent is especially delicate for
opium alkaloids, detecting even traces of morphine and codeine
(0.05 milligram), as well as of papaverine (o.i milligram).
Selenious-sulphuric acid gives the following color reactions
with the commoner opium alkaloids:
1 Mecke, Zeitschrift fiir offentliche Chemie 5, 350 (1899) and Zeitschrift fur
-analytische Chemie 39, 468 (1900).
POISONS NOT IN THE THREE MAIN GROUPS
215
Cold
Hot
Morphine
Apomorphine
Codeine
Narceine
Narcotine
Blue; then permanent blue-
green to olive-green.
Dark blue-violet.
Blue quickly changing to
emerald-green and later to
permanent olive-green.
Faint greenish yellow; then
violet.
Greenish steel- blue* later
Brown.
Gradually dark brown.
Steel-blue; then brown.
Dark violet.
Papaverine
Thebaine
cherry-red.
Greenish, dark steel-blue;
then deep violet.
Deep orange gradually fad-
ing.
Intense dark violet.
Dark brown.
PAPAVERINE
Papaverine, C2oH2iNO4, constitutes about 0.5-1 per cent, of opium. When
crude it is usually mixed with narcotine. To remove the latter, prepare the acid
H H
C C
H 3 C.O C C CH
'i i-
H 3 C.O (
Y
CH 2
oxalate of papaverine which dissolves with
difficulty in water. Crystallize this salt from
boiling water until it dissolves in concentrated
sulphuric acid without color. Convert papa-
verine oxalate into the hydrochloride by treat-
ment with calcium chloride and then liberate
the alkaloid with ammonia. This product
crystallized from alcohol is pure papaverine.
Papaverine crystallizes in colorless prisms
melting at 147. This alkaloid is insoluble in
water; soluble with difficulty in ether (i 1260),
cold alcohol and benzene; but freely soluble in
alcohol, acetone and chloroform. These
solutions are neutral, not bitter, and optically
inactive. Papaverine is a weak base which
dissolves in but does not neutralize acetic
acid. Ether partially extracts it from an
aqueous tartaric acid solution and completely extracts it from alkaline solution.
Consequently this alkaloid appears in the Stas-Otto process in ether extract B.
Chloroform extracts papaverine with almost as much ease from an acid solution
as from one that is alkaline.
Constitution. Papaverine is a monacid, tertiary base which combines with
alkyl iodides forming crystalline addition products. As it forms no acetyl deriva-
HC CH
HC C O.
V
r\c*tj
UCrla
O.CH 3
216
DETECTION OF POISONS
tive with acetic anhydride, free hydroxyl is not present. But there are probably
four methoxyl groups, for it loses four methyl groups when treated with hydriodic
acid according to Zeisel's method. Consequently all the oxygen atoms in papa-
verine are present as methoxyl groups. The researches of Guido Goldschmiedt,
extending from 1883 to 1898, have completely explained the constitution of
papaverine. Moderate oxidation with potassium permanganate and sulphuric
acid gives papaveraldine, C2oHi9NOB, without breaking the carbon chain. Fu-
sion with potassium hydroxide breaks the latter into nitrogen-free veratric acid
and the nitrogenous base dimethoxy-isoquinoline : l
1 Isoquinoline (II) is isomeric with quinoline (I) and like the latter is a monacid,
tertiary base:
I.
H H
C C
^\ /\
HC C CH
CH
II.
Hi i
C N
H
Quinoline
H H
C C
H H
Isoquinoline
H 3 CO C C CH
H 3 CO C C N
C C
H I I
H 3 CO C'
CH
I II
CO + OK
HC CH
Hi
C OCH 3
C
OCH 3
Papaveraldine
H 3 CO C C N
Y Y
H H
Dimethoxy-isoquinoline
COOK
i
H/\H
HC C OCH 3
V
c
OCH 3
Veratric acid
Detection of Papaverine
^The following general reagents precipitate papaverine in a
dilution of i : 10,000: phospho-molybdic acid, potassium bis-
muthous iodide and iodo-potassium iodide.
The following still give precipitates in a dilution of i : 5000:
tannic acid, gold chloride and potassium mercuric iodide.
POISONS NOT IN THE THREE MAIN GROUPS 217
The following special tests should be made :
1. Concentrated Sulphuric Acid. The cold colorless solution
of papaverine in this acid becomes dark violet upon gentle
warming. But even a cold solution of impure papaverine in
this acid is violet.
2. Froehde's Test. The solution of pure papaverine in this
reagent is green. Blue, violet and finally a brilliant cherry-red
color appear upon warming the solution.
3. L. E. Warren's 1 Test. Crush a very small crystal of
potassium permanganate with a glass rod and intimately mix
about 0.0005 gram of papaverine with the powder. Stir this
mixture into about 0.2 cc. of Marquis' reagent. A green color,
almost instantly changing to blue, appears. The latter color
deepens into an intense violet-blue which after some time be-
comes bluish green, green and finally a dirty brown.
Of thirty-nine alkaloids tested, the only one in any way simulating papaverine
was an unnamed alkaloid separated from sanguinaria.
4. Solutions of this alkaloid in concentrated nitric acid, or
Erdmann's reagent, are dark red.
. Heat to boiling a solution of i part of papaverine with 10 parts of nitric acid
(sp. gr. i. 06 = 10 per cent. HNO 3 ). As the solution cools, yellow crystals of
the nitrate of nitro-papaverine, C2oH 2 o(NO 2 )NO4.HNO3.H 2 O, appear. Yellow
prisms of nitro-papaverine, C2oH2o(NO2)NO4.H2O, may be obtained from this
nitrate by means of ammonia.
5. Ammonia colors the greenish solution of papaverine in
chlorine water deep red-brown which becomes later almost
black-brown.
6. Selenious -Sulphuric Acid Test. See page 215 for the
color changes given by pure papaverine dissolved in this
reagent.
PILOCARPINE
Pilocarpine, C n Hi6N 2 O2, occurs with isopilocarpine and probably also with
pilocarpidine in the leaves of jaborandum (Pilocarpus pennatifolius 2 ). The
1 Journal of the American Chemical Society 37, 2402 (1915).
2 According to Jowett, jaborine, which has been described as another alkaloid
peculiar to jaborandum leaves, is a mixture of isopilocarpine, pilocarpidine, a
little pilocarpine and pigment.
218 DETECTION OF POISONS
H O free base as usually obtained is semi-liquid, viscous, non-
C 2 H 6 C C volatile and alkaline. It dissolves but slightly in water;
No is freely soluble in alcohol, ether and chloroform; and
rip Q' insoluble in benzene. Solutions of pilocarpine and its salts
| HZ are dextro-rotatory. This alkaloid is a strong base neu-
CH2 tralizing acids and forming salts that are usually crys-
P 3 talline. Caustic alkalies, added to concentrated solutions
Q -^ of pilocarpine salts, precipitate the free base which redis-
II ^CH solves "* an excess f tne precipitant. Solutions of
If # sodium hydroxide, or sodium ethylate (C 2 H 5 .ONa), cause
a molecular rearrangement of pilocarpine. This reaction
runs more smoothly, if pilocarpine hydrochloride is heated for half an hour at
200. The product of this change is isopilocarpine, CnHi6N2O2, isomeric
and very likely stereo-isomeric with pilocarpine. Both isomeric pilocarpines
differ in melting-points, solubilities and particularly in specific rotation.
Isopilocarpine is less dextro-rotatory than pilocarpine and crystallizes in
deliquescent prisms easily soluble in water and alcohol. The salts of the two
bases also show similar differences:
Pilocarpine nitrate, CiiHi 6 N 2 O2.HNO3; mpt. 178; []D = + 82.90.
Isopilocarpine nitrate, CnHi 6 N 2 O 2 .HNOa; mpt. 159; [a]D = + 35-68.
Jowett 1 has succeeded in converting isopilocarpine into pilocarpine by means
of the same reagent used in converting pilocarpine into isopilocarpine. Pure
isopilocarpine, heated with pure alcoholic potassium hydroxide, gives a mixture
of unaltered isopilocarpine and pilocarpine. The identity of the latter with
pure pilocarpine was established by preparing the hydrochloride and nitrate
(mpt. 178). This reciprocal conversion of one alkaloid into the other strongly
supports the idea of the stereo-isomerism of pilocarpine and isopilocarpine.
Pinner was the first to show that the two nitrogen atoms of the two isomeric
bases belong to a glyoxaline ring. 2 In 1905 Jowett proposed for pilocarpine
and isopilocarpine the following formulae :
C 2 H 5 .CH CH CH 2 C N CH 3 C 2 H 6 .CH CH CH 2 C N CH 3
\ CH \ CH
OC CH 2 HC-N X OC CH 2 HC ^
V V
Pilocarpine Isopilocarpine
1 Proceedings of the Chemical Society 21, 172 (1905).
2 Glyoxaline, or imidazole (C 3 H 4 N 2 ), is obtained by the action of ammonia
upon glyoxal in presence of formaldehyde. It is a strong base and crystalline
: = jp
"I5 INH
H
h C =
HC NH
II >
HC-N
Glyoxaline
: = p
H 2 jN|H HO!
Formaldehyde
POISONS NOT IN THE THREE MAIN GROUPS 219
Detection of Pilocarpine
Ether, chloroform or benzene extracts pilocarpine from aque-
ous solutions alkaline with sodium hydroxide or carbonate.
Evaporation of these solutions leaves a thick, non-crystalline,
alkaline syrup. The general reagents especially delicate for
pilocarpine are: iodo-potassium iodide, phospho-molybdic acid,
phospho-tungstic acid and potassium bismuthous iodide.
1 . Place a particle of potassium dichromate and 1-2 cc.- of
chloroform in a test-tube. Then add pilocarpine itself, or its
solution and i cc. of 3 per cent, hydrogen peroxide. Shake for
several minutes. The mixture yellowish at first gradually
darkens and in 5 minutes is dark brown. Depending upon the
amount of pilocarpine, the chloroform is blue-violet, dark or
indigo-blue. But the upper aqueous solution gradually fades.
The chloroform mixture is an intense blue from o.oi gram of
pilocarpine and blue-violet from o.ooi gram and less. The
color lasts from an hour to a day (H. Helch 1 ).
Apomorphine (o.oi gram) colors chloroform blue- violet even without hydrogen
peroxide. Strychnine gives a barely perceptible bluish tint which changes com-
pletely within a few minutes. There is a color with antipyrine only after
acidification of the hydrogen peroxide.
2. Mandelin's reagent dissolves pilocarpine with a golden
yellow color which gradually changes to bright green and finally
to light brown.
3. The solution of pilocarpine in formalin-sulphuric acid
becomes yellow, yellowish brown and blood-red, if warmed.
Thus far fatal poisonings from this alkaloid have not occurred
and nothing is known as to the possibility of its detection in the
cadaver.
PTOMAINES
Ptomaines are basic substances containing nitrogen and may be toxic or
non-toxic. They are produced during putrefaction 'of cadavers under the in-
fluence of bacteria. They are to be regarded to some extent as products of
bacterial metabolism and are nearly always present in cadavers, especially in
those parts which are in an advanced state of putrefaction. Many ptomaines
closely resemble alkaloids. Like alkaloids they give precipitates with the general
reagents, and certain ptomaines resemble well-defined alkaloids even with special
1 Pharmazeutische Post 35, 289, 498 (1902) and 39, 313 (1906).
220 DETECTION OF POISONS
reagents. Hence ptomaines are of very great importance in forensic chemistry,
since their presence may easily lead to mistakes and false conclusions. These
putrefactive products also resemble vegetable bases in their behavior with sol-
vents. Ether extracts some of them from acid solution and others from alkaline
solution, whereas certain ptomaines are removed from alkaline solution only by
amyl alcohol or chloroform. Most of the ptomaines are strong reducing agents,
for example, they will immediately convert potasium ferricyanide into ferro-
cyanide. Consequently, they give the Prucsian blue test with a dilute mixture
of solutions of ferric chloride and potassium ferricyanide. Many of the alkaloids
like morphine resemble the ptomaines in this respect.
The resemblance of a ptomaine to a definite vegetable base is frequently con-
fined to some one reaction, and never extends to all the reactions characteristic
of the particular alkaloid. In a legal-chemical investigation no precaution,
which guards against mistaking a ptomaine for a vegetable base, should be
omitted. It is an invariable rule to make every test characteristic of the sus-
pected alkaloid, and not to be satisfied with possibly one positive test. A
determination of the physiological action of the substance should supplement
the chemical examination. A ptomaine may resemble a vegetable base chemi-
cally, and yet the two substances may differ very decidedly in physiological action.
Thus far, ptomaines have been found which show certain resemblances to coniine,
nicotine, strychnine, codeine, veratrine, delphinine, atropine, hyoscyamine,
morphine and narceine. Selmi has described a putrefactive product which
resembles morphine. Ether failed to extract it, either from asid or alkaline
solution, whereas amyl alcohol removed it with ease from an alkaline or am-
moniacal solution. It liberated iodine from iodic acid, but failed to give the
tests which are characteristic of morphine alone, namely, Husemann's, Pellagri's
and the ferric chloride tests !
The object in such cases must be to get a result about which there can be no
doubt. Every possible means must be used to isolate the alkaloid in a perfectly
pure state. When this can be accomplished, the nature of the poison can always
be established beyond question.
SAPONmS
The term saponins, or sap6nin substances, includes a large number of gluco-
side-like bodies of widespread occurrence in the vegetable kingdom and having
in common certain chemical, physical and especially physiological properties.
Their aqueous solutions when shaken foam readily. In this respect they re-
semble the soaps. Many saponin substances have a sharp, harsh taste. In
powdered form they excite violent sneezing. They are capable of holding many
finely divided substances in a state of emulsion. They dialyze incompletely
and salts precipitate them from solution. Excepting the gluco-alkaloid solanine,
which contains nitrogen and is alkaline, the saponins may be classified chemically
as nitrogen-free glucosides. Most saponins are neutral and only a few are
faintly acid. Neutral saponins and alkali salts of acid saponin substances dis-
solve in water and hot aqueous alcohol but are insoluble in absolute alcohol and
ether. Barium hydroxide and lead acetate (neutral and basic) precipitate
saponins from concentrated aqueous solution. The former gives baryta saponins.
POISONS NOT IN THE THREE MAIN GROUPS 221
Basic lead acetate precipitates all saponins but the neutral salt precipitates
only acid saponins. Ammonium sulphate is capable of salting saponin sub-
stances from solution as it does proteins. Solutions of saponins in concentrated
sulphuric acid are yellow, gradually becoming red and sometimes violet and
blue-green. The detection thus far of saponin substances in more than 50 plant
families having over 200 monocotyledenous and dicotyledenous species shows
the wide occurrence of these substances in the vegetable kingdom. Saponins
occur in roots (Senega, Saponaria), tub'ers (Cyclamen), barks (Quillaja, Guaia-
cum), fruits (Sapindus, Saponaria), seeds (yEsculus, Agrostemma. Thea), stems
(Dulcamara) and leaves (Guaiacum). In fact almost any part of the plant
organism may contain saponins. The plant families, producing saponin sub-
stances in greater abundance, are the sapindaceae, caryoph'yllaceae, colchicaceae
polygalaceae, sileneas and solanaceae. Quite considerable quantities of saponins
may occur in the particular part of the plant.
Saponin solutions, heated with dilute hydrochloric or sulphuric acid, are
hydrolyzed into sugars and a non-toxic substance insoluble in water called
sapogenin. The sapogenins have not been extensively investigated but they
are not entirely identical.
The following saponins have been more closely studied:
Digitonin: in the seeds of Digitalis pupurea.
Saponin: in the root of Saponaria offkinalis (4-5 per cent.).
Githagin: in the seeds of the corn cockle, Agrostemma githago (6 .5 per
cent.).
Senegin: in Senega root, the root of Polygala senega.
Struthiin: in levantine soap root, the root of Gypsophila struthium (14 per
cent.).
Quillaja-Sapotoxin: in the bark of Quillaja saponaria (8.8 per cent.).
Sapindus-Sapotoxin: in the fruit of Sapindus saponaria.
Sarsaparilla-Saponin: in the sarsaparilla root/the root of various kinds of
smilax.
Physiological Action of Saponins. Almost without exception saponin sub-
stances are highly toxic, if introduced directly into the blood. Most saponins
are absorbed with difficulty. Consequently healthy individuals may take dilute
saponin solutions by the mouth in considerable quantities without ill effects.
Toxic saponins act in common as protoplasmic irritants. In larger doses saponin
substances kill protoplasm. They manifest in various ways their power of acting
as protoplasmic poisons. Saponins act upon blood-corpuscles for the same
reason. R. Robert and his collaborators have shown defibrinated blood, diluted
100 times with physiological salt solution (see below), to be the best and most
convenient reagent for saponin substances. Saponins cause haemolysis and the
blood solution becomes laky. Agglutination and formation of methsemoglobin
do not occur. The freer the blood is of serum, the more pronounced the hae-
molytic action of saponin substances upon blood-corpuscles. Recent investi-
gations have shown that saponins act more vigorously upon blood-corpuscles
isolated from serum, because blood serum contains cholesterin which has a pro-
tective influence and retards haemolysis. Most likely the haemolytic action of
saponins is due to removal of cell membrane lecithin, the chief 'constituent of the
cell wall, from red blood-corpuscles, for lecithin-saponins are formed. Saponins
222 DETECTION OF POISONS
also combine with cholesterin, as well as with lecithin, forming cholesterin-
saponins. The affinities of a saponin having been satisfied by cholesterin, it no
longer acts upon the lecithin of the membrane of blood-corpuscles. Thus
cholesterin prevents haemolysis, which a saponin may produce, and so acts as
an antidote to saponin substances. Ransam 1 has made the important discovery
that addition of cholesterin checks the solvent action of a saponin upon blood-
corpuscles. At first it was not known whether this antidotal action was due to a
chemical reaction, or to absorption, that is to say, to a physical process. R.
Kobert 2 as well as Madsen and Noguchi 3 were able to dissolve cholesterin,
which is insoluble in water, in an aqueous saponin solution. They assumed that
this physiologically inactive solution contained a labile saponin-cholesterin
compound no longer having haemolytic power. Recently A. Windhaus 4 has
definitely proved that saponin-cholesterides exist. Digitonin-cholesteride,
C5sH94O28.C27H46O, crystallizes in fine needles, when a hot alcoholic solution of
digitonin (i molecule) is poured into a similar solution of cholesterin (i mole-
cule). This cholesteride is formed without elimination of water. Hence in this
reaction between digitonin and cholesterin we are dealing most probably with the
formation of a molecular compound.
Saponin solutions also dissolve white blood-corpuscles but only at higher
concentrations. A physiological action characteristic of many saponins is
exhibited in the stupefaction and killing of fish, even in water containing only
1:200,000 of saponin substance (R. Kobert).
Detection of Saponins
The matter of solubility is especially important in isolating
saponin substances from mixtures. All saponins are soluble in
water and some in alcohol, but they are practically insoluble
in ether, benzene, chloroform and petroleum ether. Employ
neutral or basic lead acetate (see above) in isolating saponins.
Decompose the washed precipitate with hydrogen sulphide,
filter and evaporate the nitrate upon the water-bath. Pre-
cipitate the saponin with absolute alcohol and ether from the
concentrated solution. Solutions of most saponins in con-
centrated sulphuric acid are red or yellowish red, gradually
becoming violet. Saponin substances give various colors with
Froehde's reagent and vanadic-sulphuric acid: brown, red-
brown, blue, green and violet (see solanin). A saponin solu-
tion heated with dilute hydrochloric acid, undergoes hydrolysis
1 Deutsche medizinische Wochenschrift 1901, 194.
2 R. Kobert, Die Saponine, Stuttgart, 1904.
Chemisches Zentralblatt, 1905, I, 1265.
4 Berichte der Deutschen chemischen Gesellschaft 42, 238 (1909).
POISONS NOT IN THE THREE MAIN GROUPS 223
and then, owing to formation of sugar, reduces Fehling's solu-
tion with heat.
Detection in Foaming Beverages (Beer, Wine, Effervescing Lemonade) l
Treat the beverage to be tested for saponin with excess of
basic magnesium carbonate, evaporate to about 100 cc. and mix
with 2 volumes of 96 per cent, alcohol. Filter after 30 min-
utes and evaporate the alcohol from the nitrate. Filter the
residue hot and extract the cold nitrate with sufficient liquid
carbolic acid 2 to leave about 5 cc. undissolved. Add ammonium
sulphate to hasten the separation of the carbolic acid layer.
Then shake the latter with water and a mixture of 2 volumes of
ether and i volume of petroleum ether. Evaporate the aqueous
solution to dryness upon the water-bath. Wash the residue
with cold absolute alcohol, in case of wine, and with acetone, in
case of beer. The residue fails to give the saponin reaction
well, that is to say, a red color with concentrated sulphuric acid,
unless treated as described. E. Schaer dissolves the residue
in concentrated aqueous chloral hydrate solution and adds the
latter to concentrated sulphuric acid as an upper layer. A
saponin produces a yellow, then purple-red and finally mallow-
blue zone.
Detection of Githagin (Corn Cockle Saponin) in Flour
Heat 500 grams of flour with i liter of alcohol (sp. gr. 0.8496
= 85 per cent, by volume). Filter hot, distil most of the alco-
hol, add absolute alcohol as well as ether to the residue and let
stand 12-24 hours. Collect the precipitate upon a filter and
dry for a short time at 100 to coagulate possible protein.
Dissolve in a little cold water, filter and precipitate githagin
from the filtrate with absolute alcohol, best with addition of
ether. Githagin thus obtained is a yellowish white powder
having a sharp, harsh taste.
To prove the presence of a saponin substance, agitate its
1 K. Brunner, Zeitschrift fur Untersuchung der Nahrungs- und Genussmittel
5, 1197 (1902).
2 Acidum carbolicum liquefactum of the German Pharmacopoeia.
224 DETECTION OF POISONS
aqueous solution which should foam. Then heat the solution
with dilute hydrochloric acid and test its reducing power with
Fehling's solution. Finally, if possible, perform the physio-
logical test with blood. Dilute defibrinated ox blood with 100
volumes of 0.9 per cent, sodium chloride solution and add the
solution of supposed githagin in 0.9 per cent, sodium chloride
solution. The blood solution at once becomes laky, if githagin
is present. According to J. Brandl, 1 Agrostemma-Sapo toxin
(githagin) produces haemolysis in very great dilution (1:50,000).
But after previous treatment with cholesterin, even o.oi gram
shows no haemolytic action whatever.
Physiological Salt Solution and Haemolysis
To prevent red blood-corpuscles from changing volume in
experiments requiring dilution of blood, an isotonic salt solution
must be used. What is an isotonic solution? If n gram-
molecules of a body A are dissolved in a definite volume of
solvent and n gram-molecules of a body B are dissolved in an
equal volume of the same solvent, certain properties of the
original solvent are changed equally in both cases. The freezing-
point of the solutions is lowered and the boiling-point raised
equally. The two solutions have the same vapor tension and
the same osmotic pressure. In other words they are isotonic.
Blood-corpuscles retain their volume unchanged, if brought
into a salt solution having the same osmotic pressure as the
blood serum. Such a salt solution is isotonic with blood serum.
In the case of human and mammalian blood an isotonic solution
of sodium chloride has a concentration of 9 per thousand =
physiological salt solution. Such a solution formerly contained
0.6 per cent, of sodium chloride. Blood-corpuscles give up
water to solutions of higher concentration than 0.9 per cent.
NaCl (hyperisotonic solutions) until osmotic equilibrium is
established. They shrivel and hence have a smaller volume.
On the other hand, blood-corpuscles in salt solutions of lower
concentration (hypisotonic solutions) take up water and be-
come distended. In diluting blood with water, this swelling
1 Archiv fur experimentelle Pathologic und Pharmakologie, 54, 245.
POISONS NOT IN THE THREE MAIN GROUPS 225
may go far enough to cause haemoglobin to separate from the
stroma and pass into the aqueous solution. This process is
called haemolysis. Alternate freezing and thawing of blood may
produce haemolysis. Various chemical substances, which act as
protoplasmic poisons, cause the same result. Such substances
are ether, alcohol, chloroform, alkalies, gallic acids, solanine,
etc. The saponins described above are also powerful haemolytic
agents. Finally, those globulicidal substances, or. haemolysins,
normally occurring in blood sera, as well as those produced in
immunization, belong in this class.
SOLANINE
Solanine, Co^HgsNOis, at the same time an alkaloid and a glucoside (gluco-
alkaloid) occurs in the potato plant (Solanum tuberosum) and in other Solanaceae
as Solanum nigrum, Solanum dulcamara and Solanum lycopersicum (tomato).
It has been found also in Scopoliaceae, as in Scopolia orientalis and Scopolia
atropoides. Solanine is not uniformly distributed in all parts of the potato plant
but is most abundant in the berry-like fruit and in the chlorophyll-free sprouts
appearing in the spring upon potatoes that lie in a cellar. Schmiedeberg and
Meyer found 0.024 gram of solanine per kilogram of peeled potatoes in January
and February but 0.044 gram in unpeeled potatoes. Potato peelings gave 0.71
gram of solanine per kilogram and potato sprouts i cm. long even 5.0 grams.
The appearance of solanine according to R. Werk is due to the life processes of
Bacterium solaniferum (?).
Solanine crystallizes in white needles having a bitter taste and melting at
244. Even boiling water dissolves only a little of this alkaloid (about i : 8000).
It is soluble in 500 parts of cold and 125 parts of boiling alcohol; and in about
4000 parts of ether. These solutions are faintly alkaline. Hot saturated solu-
tions of solanine in alcohol and amyl alcohol gelatinize upon cooling. Ether,
chloroform and benzene do not extract solanine either from acid or alkaline
solution. But hot amyl alcohol extracts solanine from acid solution and from
solutions alkaline with sodium hydroxide or ammonia. Solanine is a weak
base, readily dissolving in acids, as acetic acid, and forming crystalline salts.
Dilute hydrochloric or sulphuric acid hydrolyzes solanine to solanidine, C^Hsi-
N0 2 , galactose and rhamnose. Hydrolysis is very slow in the cold but rapid
upon heating. The hydrochloride or sulphate of solanidine separates as a diffi-
cultly soluble, crystalline powder. A good yield of solanidine is obtained,
according to Wittmann, by heating solanine under a return-condenser with 10
times the quantity of 2 per cent, sulphuric acid, until the liquid is yellowish
and the nitrate upon further boiling no longer deposits solanidine sulphate.
Solanidine, precipitated from its sulphate with ammonia and recrystallized from
ether, forms colorless, silky needles, melting at 207 and dissolving with difficulty
in water but readily in ether or hot alcohol. Solanidine is a stronger base than
solanine and the salts it forms with acids are usually crystalline and difficultly
15
226 DETECTION OF POISONS
soluble in water. Solanine and solanidine are highly toxic substances having
an action similar to that of the saponin substances (see above).
Toxic Action. Solanine taken internally is usually very imperfectly ab-
sorbed. As a glucoside its action is local and as a saponin-like substance strongly
hsemolytic, rendering the blood laky. A solanine solution even in a dilution
of i : 8300 causes complete haemolysis. Internal administration of solanine usu-
ally produces emesis and larger doses cause gastro-enteritis (gastro-intestinal
catarrh). The latter also follows intravenous and subcutaneous injection of
doses not rapidly fatal. At the same time a hsemoglobinuria may appear. (R.
Kobert, Intoxikationen.)
Detection of Solanine and Solanidine
Since very dilute mineral acids hydrolyze solanine, these acids
cannot be used to detect this alkaloid. E. Schmidt 1 suggests
the following procedure. Extract the material with cold water
containing tartaric acid. Neutralize the filtered extract with
calcined magnesia and evaporate to dryness upon the water-
bath. Extract the residue with alcohol and filter hot. Tf the
quantity of solanine is not too small, the alcoholic extract gel-
atinizes upon cooling. Otherwise, evaporate the alcoholic
solution and examine the residue for solanine. L. Kobert
extracts solanine from alkaline solution with isobutyl alcohol.
Phospho-molybdic acid is the only general reagent giving a pre-
cipitate with a solanine solution and that is yellow. But sol-
anidine, that is to say, a solanine solution that has been boiled
with excess of hydrochloric acid, being a stronger base, gives
precipitates with most of the other general reagents.
Special Tests for Solanine and Solanidine
1. A solution of, solanine in selenic-sulphuric acid 2 is rasp-
berry-red. Gentle heat favors the appearance of this color.
Solanidine gives the same result.
2. Solutions of solanine and solanidine in vanadic-sulphuric
acid 3 are orange-yellow, soon becoming red and finally blue-
violet. Solanine may be dissolved first in sulphuric acid and a
drop of vanadic-sulphuric acid added to this solution.
1 Pharmazeutische Chemie, Organischer Teil.
2 A mixture of 1.3 grams of sodium selenate (Na 2 SeO 4 .io H 2 O), 8 cc. of water
and 6 cc. of concentrated sulphuric acid.
3 Dissolve o.i gram of ammonium vanadate (H 4 N.VO 3 ) in 100 grams of con-
centrated sulphuric acid.
POISONS NOT IN THE THREE MAIN GROUPS 227
3. Solutions of solanine and solanidine in ethyl sulphuric-
acid 1 are red. An alcoholic solution of solanine, carefully
added to concentrated sulphuric acid as an upper layer, produces
a red zone where the two liquids meet (E. Schmidt).
4. A solution of solanine in concentrated sulphuric acid is
orange but becomes brownish red on longer standing or gentle
warming. Red streaks appear, if bromine water is added
drop by drop to a solution of solanine in concentrated sulphuric
acid.
5. A solution of solanine in Froehde's reagent is first yellow-
ish red then evanescent cherry-red and finally red-brown.
The methods for estimating solanine quantitatively in pota-
toes are described in Chapter VI (see page 291).
THEBAINE
Thebaine, Ci 9 H 2 iNO 3 = CnHi 5 (OCH 3 ) 2 NO, constitutes about 0.15 per cent.
of opium. This alkaloid crystallizes from dilute alcohol in leaflets having a
jj jj silvery glitter and from absolute alcohol in
C C N.CH 3 prisms melting at 193. It is nearly insoluble
-/\ /\ /\ in water, rather easily soluble in hot alcohol,
H f ff C ? C ? H2 ether> benzene and chloroform. It differs from
CH 3 O C C C CH 2 morphine in being nearly insoluble in caustic
\/ \# \/ alkalies. Its solutions are tasteless and laevo-
C C CH rotatory.
1 I H I H Constitution. Thebaine is a strong tertiary
\/> base, forming as a rule well crystallized salts
C with acids. But excess of acid, especially
mineral acid, usually decomposes these salts
R Pschorr's formula wlt ^ ease - Being a tertiary base, it easily
combines with methyl iodide, forming thebaine
iodomethylate, Ci 9 H 2 iNO 3 .CH 3 I, crystallizing in prisms. Two of the three
oxygen atoms in thebaine are methoxyl-groups ( OCHs) and the third prob-
ably forms an ether-like combination, a so-called bridge-oxygen. The thebaine
molecule appears not to contain hydroxyl.
Heated with acetic anhydride, thebaine gives the acetyl derivative of the
phenol thebaol, Ci6H 14 O 3 , and a nitrogenous product, methyl-oxy-ethylamine,
CH 3 .NH.CH 2 .CH 2 .OH. R. Pschorr has synthesized thebaol, or the methyl
ether of thebaol, and shown by this synthesis that thebaol is 3,6-dimethoxy-
4-oxy-phenanthrene (see below). Pschorr assigns to thebaine the structural
formula given above which is analogous to that of apomorphine and of morphine
(see pages 127 and 131). Thebaol has the following structural formula:
1 Add 6 cc. of concentrated sulphuric acid to 9 cc. of absolute alcohol.
228 DETECTION OF POISONS
/\ /\
HC C CH
C C CH
(4) HO
1 Hi L
Y
OCH 3 (6)
Detection of Thebaine
Ether and chloroform extract thebaine from an alkaline
aqueous solution and consequently this alkaloid appears in
ether extract B, if the Stas-Otto procedure is followed. The
general reagents, phospho-tungstic acid, iodo-potassium iodide,
potassium mercuric iodide and potassium bismuthous iodide
precipitate thebaine even from very dilute solutions. Thebaine
gives the following color reactions :
1. Concentrated Sulphuric Acid gives a deep red color with
thebaine and the solution gradually becomes yellowish red.
Froehde's reagent gives the same result.
2. Concentrated Nitric Acid dissolves thebaine with a yellow
color. With Erdmann's reagent the color varies from dark red
to orange.
3. Chlorine Water dissolves thebaine and ammonia turns the
solution an intense red-brown.
Toxalbumins
Toxalbumins are toxic, protein-like substances either already formed in the
plant or animal organism, or produced in the metabolism of pathogenic micro-
organisms. These substances as yet have not been isolated pure as individual
chemical compounds. The chemical and physiological properties of such
vegetable toxalbumins as abrin, ricin, robin and crotin are given as a matter of
fact by substances obtained from some particular part of the plant by a definite
method. The vegetable toxalbumins mentioned possess the common property
of clumping, agglutinating and precipitating red blood-corpuscles. Therefore
R. Robert classifies them as "vegetable agglutinines." A trace of one of these
agglutinines, added to defibrinated blood in a test-tube, causes clumping into a
mass resembling sealing-wax. Abrin, ricin and crotin also cause coagulation of
milk.
POISONS NOT IN THE THREE MAIN GROUPS 229
Abrin
This toxalbumin occurs in jequirity seeds from Abrus precatorius. Remove
the seed envelopes and extract the finely divided seeds with 4 per cent, sodium
chloride solution. Concentrate the filtered liquid in vacua and acidify with
acetic acid. Precipitate abrin from this solution by addition of sodium chloride
and finally purify by dialysis. Abrin is an amorphous, highly toxic powder not
entirely free from ash. Though abrin and ricin are alike in some respects, they
are not identical.
Ricin
This intensely toxic toxalbumin constitutes 2.8-3 P er cent, of the castor bean.
Remove the seed envelopes and subject the seeds to powerful pressure to remove
as much oil as possible. Then extract with 10 per cent, sodium chloride solution.
Saturate the filtered extract at the same time with magnesium and sodium sul-
phate and keep for some time in the cold at room temperature. Place the pre-
cipitate, which contains ricin, in a parchment paper dialyzing tube and dialyze
for several days. Finally dry the residual ricin in vacuo over sulphuric acid. Ricin
is an amorphous, highly toxic powder containing ash and easily soluble in 10 per
cent, sodium chloride solution. This- toxalbumin, dissolved in sodium chloride
solution, gives the protein reactions. Ricin possesses in high degree the power of
agglutinating blood-corpuscles. Use defibrinated blood for this test-tube experi-
ment, not diluted blood or blood mixed with physiological salt solution. Ricin,
according to Elfstrand, agglutinates the red blood-corpuscles of the guinea-pig
even in a dilution of i ; 600,000. Ricin agglutinates the blood of all mammals but
not to the same degree. Removing serum from the blood and substituting
physiological salt solution strengthens rather than weakens the agglutinating
action of ricin. The inference is that serum must have a certain anti-agglu-
tinating action. Separation of red blood-corpuscles into stroma and haemo-
globin 1 shows that ricin has not changed haemoglobin in the least. But the
stromata have been altered just as the blood-corpuscles have been.
To detect ricin in castor bean press-cake, or in feeds containing castor beans,
extract the finely divided material with physiological salt solution at room tem-
perature, filter and make the agglutination test in a test-tube with undiluted, de-
fibrinated blood and with blood diluted with physiological salt solution.
Crotin
Crotin is a substance obtained from the seeds of Croton Tiglium. Remove the
seed envelopes, express the oil and treat as described for abrin and ricin. Chem-
ically crotin is very similar to ricin. Abrin and ricin agglutinate the blood-cor-
puscles of all warm-blooded animals thus far tested but crotin does not behave
the same with all kinds of blood. (See R. Robert, Intoxikationen.)
Coagulation of Blood and Defibrinated Blood
Blood is a transparent fluid, the blood-plasma, suspended in which is a very
large number of solid particles, the red and white blood-corpuscles. Outside
1 The two principal components of blood-corpuscles are the stroma, which con-
stitutes the true protoplasm, and the intraglobular contents, the chief constituent
of which is haemoglobin.
230 DETECTION OF POISONS
the organism blood coagulates even in a few minutes after being drawn. In the
clotting of blood a very difficultly soluble protein, called fibrin, separates. If the
blood is still, the clot is a solid mass which gradually contracts and exudes a clear
liquid, usually yellow, the blood-serum. The coagulum, thus formed and en-
veloping the blood-corpuscles, is called the crassamentum (Placenta sanguinis).
But if the blood is whipped during coagulation, fibrin separates in threads. The
fluid separated from the latter is defibrinated blood which consists of blood-cor-
puscles and blood-serum. To obtain defibrinated blood, whip with twigs the
fresh blood removed from a vein and fibrin will separate on these. Or run the
fresh blood into an Erlenmeyer flask, containing iron filings, and shake vigorously
for several minutes. Fibrin is precipitated on the filings.
There are several ways to retard coagulation of blood, among which the follow-
ing may be mentioned:
1. Cool blood suddenly to low temperature.
2. Draw blood direct from the vein into a neutral salt solution, for example,
magnesium sulphate solution (i volume of salt solution and 3 volumes of blood)
and stir. This mixture of blood and salt will not coagulate for a day.
3. Add blood to sufficient dilute potassium oxalate solution to give a mixture
containing o.i per cent, of oxalate. The soluble calcium salts of the blood are
precipitated by the oxalate and the blood loses its power of coagulating.
4. To prepare a non-coagulating blood-plasma, pour blood into sodium fluoride
solution until it contains 0.3 per cent, of NaF.
CHAPTER V
SPECIAL QUALITATIVE AND QUANTITATIVE METHODS
Quantitative Estimation of Phosphorus in Phosphorated Oils
i. W. Straub's Method. Straub has found that his test 1
with dilute copper sulphate solution, recommended for the
qualitative detection of phosphorus, may also be used to deter-
mine phosphorus in a phosphorated oil. If such an oil is shaken
with 3 per cent, copper sulphate solution, there is first a brown-
ish black emulsion in which each individual oil drop is coated
with a film of copper phosphide, PCu 3 (?). After 4-5 hours
shaking, this brownish black color disappears and the mixture
separates into two layers. All the phosphorus in the oil is now
in the aqueous solution as phosphoric acid. This method has
the further advantage that the decolorization of the emulsion
serves as an indicator of the completion of the oxidation.
Procedure. Put 25 cc. of 3 per cent, copper sulphate solution
(taken as CuSO 4 .5H 2 O) in a separatory funnel. Add 5 cc. of
the phosphorated oil 2 and agitate the mixture vigorously for a
long time. If a shaking machine is available, place the mixture
in a thick-walled glass bottle with a tight glass stopper and
shake 3-5 hours, or until the original brown emulsion has dis-
appeared and become clear and bright blue. Separate the
aqueous solution in a separatory funnel and precipitate phos-
phoric acid at once by the molybdate method and finally weigh
as magnesium pyrophosphate,
1 Zeitschrift fur anorganische Chemie 35, 460 (1903).
2 To prepare a phosphorated oil suitable for such determinations, dissolve about
o. i gram of yellow phosphorus in the smallest possible quantity of warm carbon
disulphide and dilute this solution to 100 cc. with olive oil. Although carbon
disulphide does not affect the determination of phosphorus, it may be removed
by warming the phosphorated oil on the water-bath.
231
232 DETECTION OF POISONS
Remarks. The accuracy of this method is shown by the results of Straub's
determinations. Instead of 0.005 gram of phosphorus, dissolved in 5 cc. of oil,
he found 0.0047 and 0.00468 gram. Even very considerable dilutions of the
phosphorated oil do not affect the accuracy of the determination. In the case of
the more concentrated phosphorated oils, shaking with copper sulphate solution
must be kept up much longer.
2. A. Frankel's 1 and C. Stich's 2 Method. Dissolve the oil
in acetone and precipitate phosphorus with hot alcoholic silver
nitrate solution. Oxidize the phosphorus in the precipitate to
phosphoric acid and finally determine the latter in the usual
way.
Procedure. Dissolve 20-50 cc. of the phosphorated oil, as
phosphorated cod liver oil, in 100 cc. of acetone or ether and
completely precipitate with hot alcoholic silver nitrate solu-
tion. 3 First wash the precipitate of silver phosphide with
ether-acetone mixture and then with alcohol. Treat next with
hot 25 per cent, nitric acid, containing a little fuming acid.
Expel excess of nitric acid from the filtrate on the water-bath
and precipitate silver with hydrochloric acid. Finally filter
from silver chloride and determine phosphoric acid in the filtrate.
Remarks. Since sodium hypophosphite and phosphite are soluble in acetone
and also precipitated by acetone-silver nitrate, it is advisable first to extract a
test portion of the phosphorated oil with water and then test the aqueous extract
for these first oxidation products of phosphorus, hypophosphorous and phosphor-
ous acids. If they are present, all the phosphorated oil should first be extracted
with water in the same manner.
Phosphorus in phosphorated oils, especially phosphorated cod liver oil slowly
disappears. C. Stich found that a phosphorated cod liver oil, containing 0.05
per cent, of phosphorus, with the usual daily removal of 5 grams, lost in 3 weeks
OQ ly 3~5 milligrams of phosphorus. Such a decrease in the amount of phosphorus
in phosphorated oils is only of slight significance. Dilute oily solutions of phos-
phorus (i : 1000), when kept in tightly stoppered bottles and protected from light,
are constant as regards their phosphorus content for a long time, even 5-6
months. Moreover, phosphorus much diluted as vapor or in solution, is oxi-
dized with corresponding difficulty. The same is also true of phosphorus in the
animal organism. Therefore it is possible sometimes to detect free phosphorus
in the excretory organs, as the liver, even several weeks after phosphorus poisoning.
The distillation method is inapplicable in the quantitative estimation of phos-
1 Pharmazeutische Post 34, 117.
2 Pharmazeutische Zeitung 37, 500 (1902).
3 Silver nitrate dissolves in about 10 parts of alcohol.
SPECIAL QUALITATIVE AND QUANTITATIVE METHODS 233
phorus in oils, as cod liver oil, since only about 40 per cent, of the phosphorus
present is found in the receiver, even when the strongest oxidizing agent and the
best absorbent for phosphorus are used. To place the phosphorus-content of the
cod liver oil residue at the amount of the distilled phosphorus is not admissible,
because cod liver oil as such contains about 0.02 per cent, of combined phosphorus.
Special Methods for the Detection of Arsenic
Isolation of Arsenic as Arsenic Trichloride 1
. This depends upon the volatility of arsenic as chloride, AsCl 3 ,
in concentrated hydrochloric acid solution and in presence of
ferrous chloride. The latter serves (a) to reduce any arsenic
acid possibly present in the material to arsenious acid which with
concentrated hydrochloric acid then forms arsenic trichloride
03):
() H 3 AsO 4 + 2HC1 + 2 FeCl 2 = H 3 AsO 3 + H 2 O + 2FeCl 3 ,
(0) H 3 As0 3 + 3 HC1 = AsCl 3 + 3 H 2 O.
Procedure. Comminute the material and mix with very
concentrated hydrochloric acid (about 40 per cent.) until rather
thin. Then add 5 grams of 20 per cent, arsenic-free ferrous
chloride solution or saturated ferrous sulphate solution and
put the mixture into a capacious retort, the neck of which is
directed obliquely upward and connected with a Liebig cooler
by an obtuse angle tube, and carefully distil. Distil about a
third to a half of the original mixture. Dilute the distillate
with water and test for arsenic in the Marsh apparatus, using
hydrochloric acid for the evolution of hydrogen.
If a tubulated retort is used for the distillation, hydrochloric acid gas can be
passed in during distillation so that the liquid is kept saturated with this acid.
Electrolytic Detection of Arsenic
To detect arsenic electrolytically, put the liquid, as the sul-
phuric acid solution obtained according to the general procedure
which contains arsenic as arsenic acid (see page 156), or urine or
stomach contents, in a sufficiency wide U-tube with platinum
electrodes (Fig. 18). Pass the current through the liquid
acidified with sulphuric acid, and arsine, AsH 3 , together with
1 H. Beckurts, Archiv der Pharmazie 222, 653 (1884).
234
DETECTION OF POISONS
hydrogen will appear at the cathode, if the liquid contains ar-
senic. First test the hydrogen for arsenic by the Gutzeit arsenic
test (see page 1 63) . If a yellow spot appears on the paper moist-
ened with saturated silver nitrate solution, arsenic is present.
That this is actually arsenic may be shown by connecting the
U-tube as shown in the sketch with a chloride of calcium tube
and a Marsh reduction-tube ; an arsenic mirror then appears in
PIG. 1 8. Apparatus for the Electrolytic Detection of Arsenic.
the latter when heated to redness. Use a current having an
electromotive force of 7-8 volts. The electrolytic method is
especially adapted for the detection of arsenic in inorganic com-
pounds present in secretions, as the urine, but not for arsenic
in organic combination as cacodyl compounds and arrhenal.
An exception among these organic compounds of arsenic is
atoxyl, or the anilid of meta-arsenic acid, AsC^.NH.CeHs. The
arsenic being rather loosely bound is broken up by the electric
current with formation of arsine.
Destruction of Organic Matter and Detection of Arsenic
(According to A. Gautier 1 and G. Lockemann 2 )
The purpose of this method is to increase the delicacy of the Marsh-Berzelius
test for arsenic, and to exclude as far as possible sources of error connected with
1 Bulletin de la Societe chimique de Paris, 29, 639 (1903).
2 Zeitschrif t fur angewandte Chemie 18, 416, 491 (1905); also 19, 1362 (1906).
SPECIAL QUALITATIVE AND QUANTITATIVE METHODS 235
the destruction of organic matter, the precipitation of arsenic with hydrogen
sulphide and the evolution and drying of hydrogen gas. Organic matter is
destroyed without the use of hydrochloric acid, and arsenic is detected with-
out precipitation as arsenic sulphide. Lockemann recommends the following
procedure and uses finely divided meat as a test experiment:
Place 20 grams of finely chopped meat in a porcelain dish and add a few cc.
of a mixture of 10 parts of fuming nitric acid and i part of concentrated sulphuric
acid. Warm upon the water-bath. , The action of the acid mixture is so vigorous
that, even after the addition of about 5 cc., the entire mass, which puffs up con-
siderably at first, changes to a yellowish, homogeneous, thick, oily liquid. If too
much acid is added at once during warming upon the water-bath, the action may
be violent enough to cause sudden charring of the whole mass with copious evolu-
tion of smoke. Such an occurrence may result in loss of arsenic. Consequently,
it is advisable to add the acid mixture, amounting in all to about 20 cc., to the
meat in 1-2 cc. portions, not adding a fresh portion of acid until brown fumes
cease coming off. The mass is dark yellow and finally becomes brown after long
heating upon the water-bath. Stir with a concentrated aqueous solution of 20
grams of a mixture of potassium and sodium nitrate (i -f- r ) and evaporate
upon the water-bath. There remains a yellow, crystalline residue which still
contains organic matter. Gradually introduce this mixture in small portions
into a platinum crucible containing 10 grams of fused potassium and sodium
nitrate (i + i). Having added all the mixture, heat the crucible for a short time
over a free flame. Dissolve the cold nielt in water, add sulphuric acid and heat
upon the water-bath until nitrous fumes have been expelled. Test a cold solu-
tion of the residue for arsenic in the Marsh apparatus.
Lockemann formerly precipitated arsenic with aluminium hydroxide, Al(OH)j.
Add 10 cc. of a 12 per cent, solution of crystallized aluminium sulphate, AU-
(SO4)3i8H2O to the solution of the melt free from carbon dioxide and nitrous acid.
Render the solution alkaline with ammonia and heat about 30 minutes upon the
water-bath. Collect the precipitate upon a paper, wash with water containing
ammonia and dissolve in about 30 cc. of 10 per cent, sulphuric acid. Heat the
solution in a porcelain dish upon the water-bath until it no longer gives a test for
nitric acid with diphenylamine-sulphuric acid. 1 Then examine this solution for
arsenic in the modified Marsh apparatus 2 devised by Lockemann (Fig. 19).
Lockemann 's latest results have shown that ferric hydroxide is much more
effective than aluminium hydroxide as a precipitant of small quantities of arsenic.
Render the water solution of the melt (see above) slightly acid with sulphuric
acid, add a few cc. of iron alum solution, then in the cold, best after cooling with
ice, add just enough ammonia to precipitate all the iron. Filter after 30 minutes,
wash -the precipitate with cold water to remove nitrates completely, then dissolve
in dilute sulphuric acid and test the solution for arsenic in the Marsh apparatus.
Iron salts do not interfere with the delicacy of the Marsh test for arsenic.
1 Dissolve i gram of diphenylamine in 100 grams of concentrated sulphuric
acid. A drop of the liquid with a drop of this diphenylamine solution in a por-
celain dish should not give a blue color.
2 O. Pressler, 30 Bruederstrasse, Leipzig, Germany, supplies this apparatus
and also the ignition-tubes.
236
DETECTION OF POISONS
Zinc in sticks 1 and sulphuric acid are used in the preparation of hydrogen.
Copper is the best activator of zinc in the Marsh apparatus. Break the zinc
sticks into pieces weighing about 1.2-1.8 grams, place for a minute in 0.5 percent,
copper sulphate solution, wash with water, dry with filter paper and preserve
carefully in a closed bottle. This procedure does not interfere with the forma-
tion of the mirror, whereas addition of copper sulphate to the reduction flask
causes retention of arsenic. Copper sulphate used for this purpose should be
carefully purified by several recrystallizations. The basic properties of fused
and granulated calcium chloride, which are not entirely removed even by hydro-
gen chloride and carbon dioxide, make this an unsuitable drying agent for hydro-
PIG. 19. Marsh Apparatus Modified by Lockemann.
gen. Lockemann found that potassium carbonate, phosphorus pentoxide and
concentrated sulphuric acid cause a noticeable decomposition of arsine, and the
same is true of glass wool and cotton. Crystallized calcium chloride in pieces
about i cc. in volume is the best drying agent, because it is entirely indifferent
to arsine. Lockemann's special drying tube (see sketch) is adapted for the use
of this substance. Bohemian glass, having a wall thickness of i mm. and an
internal diameter of 4 mm., is used for ignition-tubes. These are drawn out in
two places to a length of 4 cm. The outer diameter of the constriction is 1.5
mm. and the inner about 0.5 mm. The reduction flask contains 4-6 pieces of
coppered zinc and about 15 cc. of 15 per cent, sulphuric acid are added from the
dropping funnel. After hydrogen has been passing through the apparatus for
30 minutes, heat is applied in front of the first constriction of the ignition-tube.
If the materials are arsenic-free after 1.5-2 hours heating, place the flame in
1 Lockemann has found Kahlbaum's stick zinc always arsenic-free. The
. same may be said of Bertha spelter from the New Jersey Zinc Company.
SPECIAL QUALITATIVE AND QUANTITATIVE METHODS 237
front of the second constriction of the ignition- tube. The solution of the iron
hydroxide precipitate, prepared as described above, is added to the reduction
flask from the dropping funnel which is washed with a little water or dilute sul-
phuric acid. In testing for very small quantities of arsenic, it is advisable to
cool the place where the mirror is deposited by keeping the cotton thread wet
(see sketch).
By means of the apparatus described Lockemann has detected even o.oooi mg.
of arsenic distinctly.
Moist air gradually oxidizes the arsenic mirror, but in an absolutely dry at-
mosphere even when exposed to light there is no change. In a closed tube con-
taining a little phosphorus pentoxide arsenic mirrors may be kept unchanged
even for months.
Glass wool, or cotton, noticeably decomposes arsine. The decomposition of
arsine in aqueous solution is also hastened by the presence of fine filamentary
bodies. This reaction is probably catalytic in character.
Electrolytic Estimation of Minute Quantities of Arsenic
(C. Mai and H. Hurt 1 )
By this method minute amounts of arsenic (fractions of a
milligram) are separated quantitatively at the cathode from an
arsenical electrolyte as arsine. The latter then reacts quanti-
tatively with silver nitrate as follows:
AsH 3 + 3H 2 O + 6AgNO 3 =H 3 AsO 3 + 6HNO 3 + 6Ag.
The advantages of the electrolytic detection of arsenic are
first the avoidance of traces of arsenic that sometimes come
from zinc in the Marsh test and second that destruction of or-
ganic matter is often unnecessary. T. E. Thorpe 2 has shown
the latter to be the case in the examination of beer worts and
malt extracts for arsenic. To reduce arsenic acid and its salts, a
few drops of zinc sulphate solution should be added to the sul-
phuric acid acting as the electrolyte. The cathode is said to
have a higher tension and the hydrogen to be very active.
Apparatus and Procedure. The apparatus used by Mai and
Hurt is shown in Fig. 20.
A is the reduction- tube and B a bulb-tube with 5-6 bulbs con-
taining o.oi n-silver nitrate solution. A and B are connected
1 Zeitschrift fur Untersuchung der Nahrungs- und Genussmittel 9, 193 (1905)
and also Pharmazeutische Zeitung, 1905.
2 Proceedings of the Chemical Society 19, 183 (1903).
238
DETECTION OF POISONS
by a small tube g containing pieces of pumice stone saturated
with an alkaline lead solution, or glass wool, to retain any traces
of hydrogen sulphide. Anode a and cathode e are lead strips
about 1-2 mm. thick. Their upper ends about 5 mm. thick
are luted into glass tubes b which pass through the stopper of the
U-tube and are tight. The dropping funnel d holds about 25 cc.
and its capillary end dips about 2 cm. into the solution to be
electrolyzed. Tube c for the escape of oxygen from the anode
chamber contains a little water.
PIG. 20. Apparatus for the Electrolytic Estimation of Arsenic.
Fill U-tube A up to the mark with 12 per cent, arsenic-free
sulphuric acid and bulb-tube B with 10 cc. of o.oi n-silver nitrate
solution. Turn on the current and keep at 2-3 amperes. If
the silver nitrate solution remains unchanged after hydrogen
has been running i hour, the lead cathode and sulphuric acid
are arsenic-free. Without stopping the current, introduce from
the dropping funnel the solution to be 'tested for arsenic, the
quantity of which should not be more than 10 cc. Add this
solution as slowly as possible and wash the last traces in with a
little water. If the solution contains arsenic, or arsenic acid,
SPECIAL QUALITATIVE AND QUANTITATIVE METHODS 239
the silver nitrate solution will become dark in a few minutes and
the reaction will be at an end in 3 hours. Pour the contents of
the bulb-tube through a small asbestos filter, wash with 3-4 cc.
of water and titrate excess of o.oi n-silver nitrate with o.oi
n-potassium sulphocyanate according to Volhard's method.
Calculation. The reaction above shows that 6 molecules of
silver nitrate correspond to i atom of arsenic (= 75). There-
fore i gram-molecule of silver nitrate = - gram-atom of arsenic
= = 12.5 grams of arsenic and 1000 cc. of o.oi n-silver
nitrate = 0.125 gram of arsenic.
Notes. Electrodes of platinum (foil or gauze) cannot be used in the electro-
lytic separation of arsenic as arsine, because either solid arsine or elementary
arsenic is formed. Mai and Hurt also found that gold, silver and tin cathodes
gave unsatisfactory results and carbon electrodes were not much better. Pure
lead alone meets all the requirements as a material for the electrodes. Oxygen
compounds of arsenic are quickly and completely reduced to gaseous arsine only
upon cathodes of absolutely pure lead. The attachment of a platinum wire to a
lead electrode was sufficient to cause incomplete reduction of arsenic compounds.
For this reason the electrodes consist of one piece of lead 1 without soldering on
wire of another metal. The best electrolyte is 12 per cent, sulphuric acid. A
stronger acid easily causes the formation of hydrogen sulphide and a weaker acid
has the disadvantage of lower conductivity and lower specific gravity. The
electrolyte should be specifically heavier than the solution to be tested to keep
the latter from passing at once to the bottom of the reduction-tube.
Mai and Hurt found the following amounts of As:
As taken As found
As 2 Os o.25mg. o.223mg.
As 2 O 3 o.iomg. o.opgmg.
As2Os o.iomg. o.io5mg.
For qualitative tests -the bulb-tube may be replaced by the
drying and ignition-tubes of the Marsh apparatus.
According to Mai and Hurt the statements of Thorpe and
Trotmann, that every solution can be electrolyzed without pre-
viously destroying organic matter, do not always hold. In the
examination of beer containing arsenic the results were fairly
satisfactory, but in the case of urine the results were far too
high.
1 Kahlbaum's purest lead.
240 DETECTION OF POISONS
Quantitative Estimation of Arsenic and Antimony by the Gutzelt Method
Using a special apparatus and paper sensitized with mercuric
chloride, Sanger and Black 1 have found that the Gutzeit test
can be employed to determine small amounts of arsenic quanti-
tatively. The process is very simple and requires only a short
time for completion. Sanger and
Riegel 2 have extended this method
to the estimation of antimony.
______^ Sensitized Paper. Paper strips 3
uniformly 4 mm. wide are sensitized
by being soaked in 5 per cent, solu-
tion of recrystallized mercuric chlor-
ide. These are dried, cut into 7 cm.
lengths and protected from light
and moisture in a stoppered bottle
containing calcium chloride, or soda
lime, covered with cotton.
Apparatus. A 30 cc. bottle (Fig.
21) for the reduction is closed by a
glass stopper provided both with a
thistle-tube, constricted to 2 mm. at
the end and extending nearly to the
bottom of the bottle, and with an
^SZJiSSS^ about *5 mm.
just above the stopper. Connected
with this exit- tube by a ground joint and at a right angle is a
tube exactly 4 mm. inside diameter and approximately 9 mm.
in length from the -bend.
Procedure. Place 3 grams of uniformly granulated zinc 5 in
the bottle and a strip of sensitized paper in the 4 mm. deposition
1 Proceedings of the American Academy of Arts and Sciences 43, 297-3 24 (1907) .
2 Ibid., 45, 21-27 (1909)-
3 A cold pressed paper made by Whatman has been found to give the best
results.
4 This all glass apparatus, suggested by Mr. W. A. Boughton, is now in use
in the Harvard laboratory and is a modification of Sanger's original apparatus.
5 Bertha spelter from the New Jersey Zinc Company, New York, has been
proved free from arsenic.
* I
l
J
5 1 15 20 25 30 35 40 50 60 70
FIG. 22. Standard Arsenic Bands in Micromilligrams of As 2 O 3 (Initial).
5 10 15 20 25 30 35 40 50 60 70
FIG. 22a. Standard Arsenic Bands in Micromilligrams of As2O 3 (Hydrochloric Acid
Development).
i
111
5 10 15 20 25 30 35 40 50 60 70
FIG. 22b. Standard Arsenic Bands in Micromilligrams of As 2 O 3 (Ammonia Development).
(Facing Page 240)
SPECIAL QUALITATIVE AND QUANTITATIVE METHODS 241
tube. In estimating arsenic, place in the enlargement of the
exit-tube a loose plug of lean absorbent cotton that has been
kept over sulphuric acid; an hour's preliminary run is necessary
to mo : sten the cotton partially. In the case of antimony sub-
stitute for cotton a disc of filter paper that has been moistened
with normal lead acetate, dried and kept in a well stoppered
bottle. Before inserting this disc moisten it with a drop of
water. Next add 15 cc. of diluted hydrochloric acid 1 (i : 6)
and let the hydrogen run 10 minutes to make sure the reagents
cause no stain. Then add the whole, or an aliquot part of the
solution to be tested. Arsenic will produce a color on the paper
in a few minutes which will reach a maximum within 30 minutes.
Antimony produces no visible effect on the sensitized paper,
unless the amount is above 70 mmgr. (= 0.070 mg.) when a
gray color may appear. If there is any color, another trial
should be made with a smaller portion of solution. In the
determination of arsenic a disc of lead acetate paper should be
inserted beneath the cotton as a precaution against the possible
formation of hydrogen sulphide.
Standard Bands. (a) Arsenic. Dissolve i gram of resub-
limed arsenious oxide in a little arsenic-free sodium hydroxide,
acidify with sulphuric acid and make up to a liter with recently
boiled water. Dilute 10 cc. of this solution (I) to a liter with
freshly boiled water which gives a solution (II) containing
o.oi mg. of arsenious oxide per cc. Using definite volumes
of solution II, measured from a burette, prepare a series of color
bands (Fig. 22), taking a fresh charge of zinc and acid for each
portion. The color ranges from lemon-yellow through orange-
yellow to reddish brown.
(b) Antimony . Dissolve 2.3060 grams of pure, recrystallized
tartar emetic in a liter of water. This solution (I) contains i .o
mg. of antimonious oxide per cc. By dilution of (I) solutions
containing 0.01 mg. (II) and o.ooi mg. (Ill) are prepared and
used in making sensitized bands.
1 Synthetic hydrochloric acid, made from electrolytic hydrogen and chlorine
by the Hooker Electrochemical Company, New York, is said to be entirely free
from arsenic. Tr.
16
242 DETECTION OF POISONS
These bands will eventually fade but they may be preserved
longer by being sealed in glass tubes in the bottom of which is
phosphorus pentoxide covered with cotton. The color of the
arsenic bands may be developed (i) by placing the band in hy-
drochloric acid (i : i) for 2 minutes at a temperature not over
60, washing thoroughly, drying and sealing as before; (2) by
treating for a few minutes with ammonium hydroxide, which
gives a dense, coal-black color, washing, drying and sealing in a
tube over quicklime.
To develop the antimony band; let it stand in a test-tube
covered with normal ammonium hydroxide 5 minutes. A
black band is slowly developed. These bands may be protected
as described, or placed between glass plates cemented together
and bound with passepartout .paper.
The more dilute standard solutions must be freshly made up
within a few hours of use.
Notes. Solutions should be as free as possible from sulphur compounds
yielding hydrogen sulphide; interfering organic matter; and metals retarding
formation of arsine and stibine. The cotton in the exit-tube should be replaced
after 10-12 runs, and the lead acetate disc after each run. If the solution
contains arseniate, reduce with 10 cc. of arsenic-free sulphurous acid and expel
the excess.
The absolute delicacy of the method is set at 0.00008 mg. of arsenious oxide
and 0.0005 m g- of antimonious oxide. The practical delicacy, using a band
4 mm. wide, is o.ooi mg. of arsenious oxide and 0.002 or 0.003 m S- f anti-
monious oxide. By using, however, a band 2 mm. wide in a correspondingly
narrow exit- tube, a practical delicacy of 0.0005 mg. of arsenious oxide and o.ooi
mg. of antimonious oxide is obtainable. In length of band and density of
developed color, the effect of arsine on the sensitized paper is from 2-3 times
as great as that of stibine. The authors do not claim a greater accuracy for the
method than within 10 per cent.
Biological Detection of Arsenic by Penicillium Brevicaule
B. Gosio 1 was the first to show that certain moulds, grown
upon media containing minute quantities of arsenic, produce
volatile arsenic compounds characterized by a garlic-like
odor. Seven species of moulds were found to have this power.
Penicillium brevicaule, however, which Gosio isolated from
^'Azione di alcune muffe sui composti fissi d'arsenico," Rivista d'igiene e
sanita publica, 1892, 201.
SPECIAL QUALITATIVE AND QUANTITATIVE METHODS 243
air, and which was first found upon decaying paper, possessed
this property in the highest degree. Gosio states that we are
justified in regarding Penicillium brevicaule as a living reagent
for arsenic. Even o.ooooi gram of arsenic can be recognized
with certainty by this biological test. The test is so delicate
that it should be of great value in toxicological analysis in the
preliminary examination for arsenic.
A. Maasen 1 states that a temperature of 28 to 32 is most
favorable to the growth of the mould. Crumbs of wheat
bread were found to make an especially good culture-medium.
When this material is used, a vigorous growth of mould is
visible even in 48 hours. Sometimes a test for arsenic can be
finished in a few hours, and always in 2 or 3 days. The char-
acteristic garlic odor from weak, arsenical cultures can be dis-
tinctly recognized even after several months. That these
"arsenic moulds" do not produce gases having a garlic odor
from sulphur, phosphorus, antimony, boron and bismuth com-
pounds, is an important fact. But Penicillium brevicaule
possesses in high degree the power of converting solid selenium
and tellurium compounds into volatile substances having a pe-
culiar odor. The odor, especially from tellurium cultures, is
like that produced by arsenic cultures, namely, distinctly like
garlic! The odor from selenium cultures, however, differs
fiom that arising from arsenic cultures. It is more of a mer-
captan odor.
Biginelli 2 found that the gases, generated from arsenic
cultures by Penicillium brevicaule, are completely absorbed
by mercuric chloride solution. Colorless crystals, having
the composition (AsH(C 2 H 5 ) 2 .2HgCl 2 ), are formed. This
is a double compound of mercuric chloride and diethyl arsine.
This compound can easily be decomposed. It then diffuses
an intense garlic odor. On the other hand, Klason 3 has recently
shown as a result of an investigation of the gas given off when
Penicillium brevicaule subsists upon an arsenical medium that
1 Arbeiten aus dem Kaiserlichen Gesundheitsamt, 1902, 478.
2 Chemisches Centralblatt (1900), II, 1067, and also (1900), II, noo.
3 Berichte der Deutschen chemischen Gesellschaft 47, 2634 (1914).
244 DETECTION OF POISONS
a double compound of ethyl-cacodylic oxide and mercuric
chloride is formed, the formula of which is (C 2 H 5 ) 2 As-O-As-
(C 2 H 5 ) 2 + 4HgCl 2 .
R. Abel and J. Buttenberg 1 state that a mould to be of
use in the biological detection of arsenic must satisfy the fol-
lowing conditions: "It must grow rapidly, and not generate
any odors during growth, except the garlic odor produced from
an arsenical medium. It must not be restricted as to culture
medium. It must grow in presence of large, or very small
quantities of arsenic. Finally, it must demonstrate its specific
action in presence of metallic arsenic and all kinds of arsenical
compounds."
The best material for these experiments is white or Graham
bread, either of which is a favorite culture medium for moulds.
The crust is the only part of bread having a specific aromatic
odor. When this has been removed, the crumbs may be said
to be practically odorless.
Procedure. When the material examined is liquid, absorb
it completely by adding bread crumbs, and scatter a small
quantity of dry bread over the surface. Solid material should
be finely ground, or cut into as small pieces as possible, and
placed in not too small a flask. Add at least the same quantity
of bread crumbs, thoroughly mix the two substances by shaking,
and moisten the mass with a little water. Close the flask with
a cotton plug, and sterilize in steam. Sterilization must kill all
micro-organisms in the flask. Therefore, heat the flask in an
autoclave 10 to 30 minutes under a pressure of i to 1.5 atmos-
pheres. There is no danger of volatilizing arsenic during ster-
ilization. Then inoculate the sterilized material when cold.
Place in a flask a sh'ce of potato, superficially coated with
mould in the spore-forming stage, and agitate it with bouillon
(peptone), salt solution or sterilized water, until it is finely
disintegrated. Observe all necessary precautions, and add
the mould, suspended in water, in sufficient quantity to
impregnate the entire surface of the material suspected of
containing arsenic. Theie should not be more liquid, how-
1 Zeitschrift fur Hygiene, 32, 440 (1899).
SPECIAL QUALITATIVE AND QUANTITATIVE METHODS 245
ever, than the culture medium will absorb. Too much mois-
ture retards the growth of the mould. Finally, draw a tight
rubber cap over the mouth of the flask and cotton plug. Flasks
thus closed may stand in the room, but it is better to keep them
at a higher temperature, for example, in an incubator at 37,
since these conditions are most favorable to the growth of
the mould. As soon as a growth of mould is distinctly visible
to the naked eye upon the medium, the first indication is given
that a test of the culture for volatile arsenic compounds may
prove successful. In a very favorable case, this is possible in
24 hours. There is always a luxuriant growth of mould in 48
to 72 hours, so that a decision can be reached. If there is no
odor, the flask is closed, and the test is repeated once or twice
daily on the following days.
Sulphuric, hydrochloric and other strong mineral acids prevent the growth
of the mould. This preventive action may be overcome by neutralization
with calcium carbonate, which may be present in excess without ill effect.
Alkalies also interfere with the growth of the mould. They may be removed
by neutralization with tartaric or citric acid, either of which may be present in
excess. The great advantage of the biological over the purely chemical method
lies in the fact that less time is required to get a result. The tedious and un-
avoidable destruction of organic matter in the material is rendered unnecessary
Moreover, a number of tests for arsenic may be made at the same tune.
Abel and Buttenberg (loc. cit.) speak as follows, regarding this method:
"The biological method of detecting arsenic has so many advantages, that it
deserves to be recommended for the most varied purposes. Its application is
very general, and the method of procedure is simple. The culture of the mould
can be kept a long time, even a year or more, without being revived. The
test is very delicate, and the odor is readily recognized. The generation of the
odor, in the case of cultures containing only o.oooi gram of arsenic, can be
demonstrated for a week."
Besides being practically unlimited in application, the biological method is
extraordinarily delicate. In this respect, it exceeds the best known chemical
methods for detecting arsenic. It is, for example, considerably more delicate
than Bettendorff's test, and it might equal in delicacy the Marsh and Gutzeit
tests.
Detection of Arsenic in Organic Arsenic Compounds
Cacodylic Acid, Arrhenal, Atoxyl 1
The ordinary reagents usually fail to show arsenic in an or-
ganic arsenic compound dissolved in water. Several of these
1 C. E. Carlson, Zeitschrift fur physiplogische Chemie 49. 4*o (1906).
246 DETECTION OF POISONS
compounds persistently resist the most powerful oxidizing and
reducing agents.
Cacodylic Acid, (CH 3 ) 2 AsO-OH, and its salts have been used
of late as drugs. A 2 per cent, solution of sodium cacodylate,
(CH3) 2 AsO-ONa.3H 2 O, conducts the electric current very
feebly but no arsine appears at the cathode. BettendorfFs
reagent (stannous chloride-hydrochloric acid) does not cause
separation of arsenic from cacodylic acid even after evaporation
with hydrochloric acid and potassium chlorate. If heated with
stannous chloride-hydrochloric acid, cacodylic acid is reduced
to the foul-smelling cacodylic oxide, [(CH 3 ) 2 As] 2 0, recognized
by its odor. Distillation of sodium cacodylate by Schneider's
method with the strongest hydrochloric acid gives no arsenic
trichloride in the distillate. The arsenic changes to another
form, not precipitable by hydrogen sulphide. Evaporation of
the distillate upon the water-bath with nitric acid leaves solid,
non-volatile cacodylic acid in which arsenic may be detected
by reduction with sodium carbonate-potassium cyanide mixture.
Even fuming nitric acid does not oxidize cacodylic acid to ar-
senious or arsenic acid.
Arrhenal, Sodium Methyl-Arseniate (CH 3 )AsO(ONa) 2 .5H 2 0,
forms white crystals very soluble in water. Possibly owing to
partial hydrolysis, an aqueous solution of this compound is
alkaline and conducts the electric current feebly. Only traces
of arsine appear at the cathode after electrolysis in presence of a
good conductor. In arrhenal the arsenic is not held as strongly
as in the cacodyl compounds. Hydrogen sulphide precipitates
yellow arsenic trisulphide. Distillation with strong hydro-
chloric acid gives arsenic trichloride in the distillate. Betten-
dorff's reagent gives a red-biown precipitate, if considerable
arrhenal is present.
Atoxyl, the Anilide of Metarsenic Acid, AsO 2 .NH.C 6 H 6 ,
forms white, odorless crystals readily soluble in water and hav-
ing a faint, saline taste. As compared with cacodylic acid,
arsenic in atoxyl is less firmly bound. Electrolysis gives arsine
abundantly at the cathode. Hydrogen sulphide precipitates
sulphide of arsenic. Arsenic trichloride passes over, upon dis-
SPECIAL QUALITATIVE AND QUANTITATIVE METHODS 247
tillation with concentrated hydrochloric acid. Bettendorffs
reagent gives a lemon-yellow precipitate.
Urine. In suspected arsenic poisoning first examine the urine, since arsenic
is very slowly eliminated by this channel. Carlson in experiments upon him-
self was able to detect arsenic direct in the urine by the electrolytic method and
also by the Gutzeit and Marsh tests. He took 10 drops of Fowler's solution 1
daily. Five days after the last dose Carlson could still get a distinct test for
arsenic in concentrated urine. The urine was not wholly free from arsenic until
14 days had passed. He then experimented with sodium cacodylate, taking
daily 29 drops of a i per cent, solution. He could not detect a trace of arsenic
in the urine by the electrolytic method. Therefore the salt of cacodylic acid
had passed through the organism unaltered. But cacodylic acid can be de-
tected easily in the urine, upon treating the latter with hypophosphorous acid
(sp. gr. i.is). 2 Cacodylic oxide is formed and can be recognized by its odor.
Sometimes the mixture must stand several hours in a closed test-tube. Arrhenal,
in daily doses of about 30 drops of i per cent, solution, behaved like the cacodyl
compound. Arsenic could not be detected in the urine by electrolysis. Con-
sequently neither arsenious nor arsenic acid had been formed within the organism.
Hypophosphorous acid immediately precipitated arsenic from arrhenal and gave
the cacodyl odor.
To detect cacodylic acid in urine, phosphorous acid, as well as zinc or tin and
hydrochloric acid, may be used instead of hypophosphorous acid. Frequently
it is advisable to oxidize most of the organic matter in the urine beforehand.
Boil 25 cc. of urine with 25 cc. of water, 5 per cent, potassium permanganate
solution and 10 cc. of 25 per cent, sodium hydroxide solution, until the filtrate
is odorless and nearly colorless. Excess of hydrochloric acid (sp. gr. 1.19) and
zinc filings, added to this nitrate, produce with heat the odor of cacodyl, if the
urine contains cacodylic acid. Arsenic from atoxyl can be isolated at the cathode
in the form of arsine by electrolysis. Therefore arsenic can be detected in the
urine electrolytically after administration of atoxyl.
Quantitative Estimation of Minute Amounts of Arsenic
(Karl Th. Morner 3 )
This method is said to be useful in estimating arsenic quantitatively in various
kinds of fabrics and in urine in cases of poisoning. It is a titration method de-
vised especially for quantities of arsenic not exceeding 0.5 mg. Arsenic is
first precipitated as trisulphide with thioacetic acid, CH 3 .CO.SH. Under the
conditions arsenious as well as arsenic acid is thus precipitated. In alkaline
1 Fowler's solution contains i per cent, of As 2 0j as potassium arsenite.
2 Instead of free hypophosphorous acid, prepare Engel and Bernard's arsenic
reagent (Comptes rend, de 1'Acad. des sciences 122, 399), or J. Bougault's (I.
Pharm. Chim. (6), 15, 527). Dissolve 20 grams of sodium hypophosphite in
20 cc. of water and add 200 cc. of hydrochloric acid (sp. gr. 1.17). Filter through
a cotton plug to remove NaCl and use the nitrate.
3 Zeitschrift fur analytische Chemie 41, 397 (i9 2 )'
248 DETECTION OF POISONS
solution potassium permanganate readily oxidizes arsenic trisulphide completely
to arsenic acid and sulphuric acid:
As 2 S 3 + 140 = As 2 5 + 3SO 3 .
Potassium permanganate solution, added to an alkaline solution of arsenic
trisulphide, immediately loses its color, being decomposed in the proportion of
9 molecules to i molecule of arsenic trisulphide. 1 Since 2 molecules of potassium
permanganate in sulphuric acid solution yield 5 atoms of oxygen for oxidation,
9 molecules 'according to the proportion
2 :$ = 9 :x (x = 22.5)
should give 22.5 atoms of oxygen. But according to the reaction above, only
14 atoms of oxygen are used to oxidize i molecule of arsenic trisulphide, whereas
the remaining 8.5 atoms are stored up in the precipitate as hydrated manganese
dioxide (MnO 2 .H 2 O). But if the reaction mixture is heated with oxalic acid in
presence of dilute sulphuric acid, these oxygen atoms become active:
(MniO O) COOH
+ I i = MnSO 4 + H 2 + 2 C0 2 + H,O.
so 4 iHsi cob ;H
Since 2 molecules of KMnO4 yield 5 atoms of oxygen and since 14 atoms of
oxygen are necessary for i molecule of As 2 S 3 , according to the following pro-
portion
Atoms : Mols.KMnO 4
5 : 2 = 14 : x (x = 5.6)
5.6 molecules of potassium permanganate are required for i molecule of As 2 Sj
(= 214), or 2 atoms of arsenic (= 150).
1000 cc. of o.oi n-potassium permanganate (= 0.3162 gram KMnO4) contain
in solution = 0.002 gram-molecule of KMnO 4 which according
10 X 10 X 10
to the proportion
Gram-mols.KMnO 4 : Grm. As
5.6 : 150 = 0.002 : x (x = 0.0536)
represents 0.0536 gram of arsenic. Hence 1000 cc. of o.oi n-potassium per-
manganate solution correspond to 0.0536 gram of arsenic.
Procedure. Dissolve arsenic trisulphide in 0.5 per cent, potassium hydroxide
solution 2 and run this solution into a small flask containing 25 cc. of o.oi n-potas-
sium permanganate solution. Mix the contents and add 5 cc. of 5 per cent, sul-
phuric acid, as well as the quantity of o.oi n-oxalic acid solution found necessary
by special titration. Warm until the color is discharged and finally titrate with
o.oi n-potassium permanganate solution.
1 Since 2 mols. KMnO 4 give in alkaline solution 3 atoms of available oxygen
(2KMnO 4 = 2MnO 2 + 30 + K 2 O), i mol. of As 2 S 3 , according to the pro-
portion: 3:2 = i4:x (x = 9.33), requires not 9 mols, but more exactly 9.33
mols. of KMnO 4 .
2 Ammonium Hydroxide cannot be substituted for potassium or sodium
hydroxide solution.
SPECIAL QUALITATIVE AND QUANTITATIVE METHODS 249
Prelimnary Titration. Add the same quantity .of 0.5 per cent, potassium
hydroxide solution used to dissolve arsenic trisulphide, as well as 5 cc. of 5 per
cent, sulphuric acid, to 25 cc. of o.oi n-potassium permanganate solution. Heat
the mixture to boiling and add oxalic acid solution in slight excess so that the
liquid becomes colorless. Titrate back with o.oi n-potassium permanganate.
This titration shows how much oxalic acid solution, in conjunction with traces
of reducing substances that may be present in the potassium hydroxide solution
or sulphuric acid, is needed in a regular titration for the exact reduction of 25
cc. of o.oi n-potassium permanganate.
Example. Suppose that 25.5 cc. of oxalic acid solution were required to
decolorize the boiling liquid. Titration required 0.3 cc. of o.oi n-potassium per-
manganate solution. Therefore 25 + 0.3 = 25.3 cc. of o.oi n-potassium per-
manganate correspond to 25.5 cc. of oxalic acid solution and 25 cc. of the former
correspond to 25.2 cc. of the latter solution. Consequently in a regular titration
25-2.cc. of o.oi n-oxalic acid solution must be used.
The amount of potassium permanganate in 25 cc. of o.oi normal solution is
sufficient for all amounts of arsenic up to 0.5 mg. Morner's method of deter-
mining arsenic gives very reliable results, if arsenic is in the form of the trisul-
phide and free from every other substance soluble in 0.5 per cent, potassium
hydroxide solution and capable of reducing permanganate.
Using the strongest hydrochloric acid, Morner first distils arsenic as arsenic
trichloride by the Schneider-Fife method. About 200 sq. cm. of carpet, 100 sq.
cm. of other woven and paper materials and 15 grams of sealing wax, stearine
of wax candles and dried apples were used for each determination of arsenic by
this method. According to Morner, the distillate from such materials by the
Schneider-Fife method always contains organic matter, even when caught in
dilute nitric acid. To remove this organic matter before precipitating arsenic
with thio-acetic acid, collect the distillate in a receiver containing dilute nitric
acid and evaporate to dryness in a porcelain dish. Add to the small residue in the
dish upon the water-bath successively 2 cc. of potassium hydroxide solution
(0.5 per cent. KOH) heating i minute, then 2 cc. of potassium permanganate
solution (5 per cent. KMnO 4 ) heating about 3 minutes, and finally i cc. of
tartaric acid solution (20 per cent. Hi.C&tOs) 1 heating until the color is dis-
charged. Filter into a porcelain dish, wash the filter with a little water and set
the dish upon a boiling water-bath. Add after i minute i cc. of thio-acetic acid
(5 per cent. CH 3 . COSH) 2 and warm the mixture 3 minutes. Arsenic is precipi-
tated as arsenic trisulphide. After cooling for 5 minutes, collect the precipitate
upon a filter and wash first 5 'times with 2 cc. portions of 0.5 per cent, sulphuric
acid and then 3 times with 2 cc. portions of water. Place under the funnel a
1 Tartaric acid readily dissolves the precipitate of manganese peroxide. To
reduce the latter, Morner used oxalic and lactic acids, sodium sulphite and also
thio-acetic acid. But tartaric acid proved to be bettter than any of these
substances.
2 Prepare thio-acetic acid solution by shaking 5 cc. of thio-acetic acid with
100 cc. of water. Filter and keep this solution in a dark flask. This solution
gradually decomposes with evolution of hydrogen sulphide:
CH 3 .COSH+H 2 =CH,.COOH+H,S.
250 DETECTION OF POISONS
small flask containing 25 cc,of o.oi n-potassium permanganate solution and pour
over the filter 3 portions of 0.5 per cent, potassium hydroxide solution, using 2 cc.
each time. The alkaline solution of arsenic trisulphide thus drops directly into
the permanganate solution. Otherwise, proceed as described. Subtract 0.3 cc.
of o.oi n-permanganate solution from the volume of this solution used. This
correction is necessary because even the finer qualities of filter paper contain
traces of substances which dissolve in 0.5 per cent, potassium hydroxide solu-
tion and reduce permanganate. 1
Note. The procedure described separates arsenic trisulphide from every other
substance soluble in 0.5 per cent, potassium hydroxide solution and capable
of reducing potassium permanganate. This method is accurate to 0.02 mg.
of arsenic.
Detection of Salicylic Acid in Foods and Beverages
Wine. 2 Place 50 cc. of wine in a cylindrical separating funnel
with 50 cc. of a mixture of equal parts of ether and petroleum
ether. Shake frequently, taking care not to form an emulsion
but yet to mix the liquids thoroughly. Remove the ether-
petroleum ether layer, pour through a dry filter, evaporate
upon the water-bath and add a few drops of ferric chloride solu-
tion to the residue which becomes red-violet if salicylic acid is
present. But if the color is black or dark brown, add a few
drops of hydrochloric acid, dissolve in water, extract with
ether-petroleum ether and proceed with the extract as just
described.
Meat and Meat Products. 3 For experimental purposes add
about o.oi gram of salicylic acid to some chopped meat. Ex-
tract the finely divided material with 50 per cent, alcohol and
add some milk of lime to the filtered alcoholic solution. Evapo-
rate to dryness upon the water-bath and stir the residue with a
slight excess of dilute sulphuric acid. Shake with ether without
filtering, pass the ether extract through a dry filter and evapo-
1 After passing through the entire process in several blank experiments,
Morner never obtained higher results for permanganate used. Consequently
the method of washing described completely removes tartaric and thio-acetic
acids.
2 "Official Directions for the Chemical Examination of Wine" of June 25th,
1896. (German.)
3 "Agreements with regard to uniformity in inspecting and testing foods,
household supplies and other articles used in the German Empire" Heft I, 36.
SPECIAL QUALITATIVE AND QUANTITATIVE METHODS 251
rate. Dissolve the residue in hot water and test the filtered
solution for salicylic acid with very dilute ferric chloride
solution.
Milk. 1 Mix 100 cc. of milk with 100 cc. of water at 60.
Precipitate with acetic acid and mercuric nitrate solution, using
8 drops of each, shake and filter. Extract the filtrate with 50
cc. of ether, evaporate the ether, dissolve the residue in 5 cc.
of hot water and test the filtered solution for salicylic acid with
dilute ferric chloride solution (sp. gr. i.oo5~i.oib).
Maltol
Maltol, CeHeOs, 2 is formed in the preparation of caramel from malt, possibly
from maltose or isomaltose. Ether or chloroform extracts this substance from
the condensed vapors given off during caramelization and also from beer-wort.
Maltol crystallizes in monoclinic prisms and plates from a cold saturated solution
in 50 per cent, alcohol (Osann). Chloroform gives denser crystals. This sub-
stance dissolves with difficulty in cold water or benzene; more readily in hot
water, alcohol, ether or chloroform; and is insoluble in petroleum ether. It dis-
solves in caustic alkaline solutions but is reprecipitated by carbon dioxide.
Maltol sublimes in shining leaflets and is volatile with water vapor. It reduces
silver solution in the cold and Fehling's solution with heat. An aqueous maltol
solution resembles salicylic acid in becoming intense violet with ferric chloride
solution, but differs from carbolic and salicylic acids in not turning red with
Millon's reagent.
Maltol, shaken with benzoyl chloride and sodium hydroxide solution, gives
a mono-benzoyl derivative and consequently must contain one hydroxyl
group.
Aqueous Chloral Hydrate Solution as a Solvent for Alkaloids, Glucosides
and Bitter Principles and Its Use in lexicological Analysis
Richard Mauch
(Communication from Professor E. Schaer's Institute, Strassburg)
One part of water at 17.5 dissolves 4 parts of chloral hydrate, forming a very
mobile solution which is easily filtered and capable of being kept for a long time
without decomposition. This 80 per cent, solution of chloral hydrate easily
dissolves relatively large quantities of alkaloids and glucosides without altering
them chemically. At 17.5 one part of each of the following substances re-
quires for solution the number of parts of solvent stated in the table:
1 Method of Ch. Girard, Zeitschrift fiir analytische Chemie 22, 277 (1883) and
the above "Agreements" Heft I, 62.
2 J. Brand, Berichte der Deutschen chemischen Gesellschaft 27,806 (1894),
H.Kiliani and M. Bazlen, Ibidem 27, 3115 (1894).
252
DETECTION OF POISONS
Chloral Hydr
Solution
(80%)
ate
Water
600
20OO
700
500O
5OOO
6600
Ether
5
freely soluble
freely soluble
1250
125
1300
Chloroform
3-5
2
freely soluble
100
4
6
6
Cocaine
... 5
Morphine
Santonin
Strychnine
5
4
6.5
6 5
Veratrine. . .
7. ?
Caffeine is the only alkaloid which forms with chloral hydrate a molecular
compound soluble in water. If a chloral hydrate solution of an alkaloid, which
has been freshly prepared in the cold, is diluted with considerable water, the
unchanged alkaloid is precipitated almost quantitatively, for instance, morphine,
strychnine and quinine. Substances like picrotoxin, santonin and acetanilide
behave similarly. But when such solutions stand for a long time at ordinary
temperatures, or are heated for 1-2 hours, chloral hydrate is decomposed by
the vegetable base into chloroform and formic acid. Since the alkaloidal salts
of formic acid are soluble in water, dilution with this solvent does not precipitate
the alkaloids. R. Mauch has shown clearly that atropine, brucine, quinine,
cocaine, morphine, narcotine, strychnine and veratrine behave as just described.
In the tests ordinarily made with the ether or chloroform residue, R. Mauch
recommends dissolving the residue in 80 or 60 per cent, chloral hydrate solution.
The "chloral solution" should prove of great value in color tests which depend
upon the use of pure sulphuric acid or sulphuric acid containing iron or molybdic
acid. These solutions contain so little water that it cannot modify the action
of sulphuric acid upon the substance in solution. Such a "chloral solution" is
also well adapted for zone tests. An aqueous solution forms an upper layer with
the "chloral solution," and the latter forms an upper layer with concentrated
sulphuric acid.
Specific gravity of 80 per cent, chloral hydrate solution = 1.514.
Specific gravity of 60 per cent, chloral hydrate solution = 1.3535.
In the tests ordinarily performed in test-tubes, it is best to use small tubes
(6 or 7 cm. high; i cm, in diameter) holding 6 cc. They should not be made of
too thin glass. The chloral solution cannot be used in detecting picrotoxin, be-
cause chloral hydrate itself produces the same reduction changes caused by
picrotoxin. The same is true of the test for strychnine, where sulphuric acid
and potassium dichromate, or any other oxidizing agent, are used. Coniine and
nicotine also belong to the class of alkaloids which cannot be detected in chloral
hydrate solution. Concentrated chloral hydrate solutions cannot be used di-
rectly in making tests with general alkaloidal reagents, because precipitates do
not appear until the solutions have been diluted with 6-8 volumes of very
dilute hydrochloric or sulphuric acid.
In using the "chloral hydrate method'' in toxicological analysis, the ether,
chloroform or amyl alcohol extract should be evaporated with gentle heat upon a
SPECIAL QUALITATIVE AND QUANTITATIVE METHODS 253
watch-glass of medium size (about 5 cm. diameter) and not too flat. Add to the
residue, depending upon the quantity, about 3 cc. of 75 per cent, chloral hydrate
solution. Cover the glass and let it stand for some time. Occasionally tilt
the glass and bring the solution thoroughly in contact with the residue. Pass the
solution through a very small filter, if necessary, and wash both watch-glass and
filter with a few drops of pure chloral hydrate solution. Use this chloral hydrate
solution for the individual tests. In testing for strychnine, evaporate a part of
the chloral solution to dryness upon the water-bath. Warm, until the residue
does not smell of chloral, and then test for strychnine with sulphuric acid and
potassium dichromate.
To recover from the chloral hydrate solution most of the alkaloids and sub-
stances like picrotoxin, acetanilide and phenacetine, add excess of sodium hy-
droxide solution and extract thoroughly with a little chloroform. The "chloral
hydrate method" is conducive to very neat work and this is a great advantage.
The use of metallic utensils like knives and spatulas is entirely unnecessary.
ESTIMATION OF ALKALOIDS
i. Picrolonate Method of H. Matthes 1
Knorr 2 gave the name picrolonic acid to i-p-nitrophenyl-3-
methyl-4-isonitro-5-pyrazolone. This compound is formed by
the action of nitric acid upon methyl-phenyl-pyrazolone.
Picrolonic acid resembles picric acid in its properties and is
characterized by forming crystalline salts with many organic
bases, as the alkaloids. As a rule these salts dissolve with
dimculty and are yellow or red. Heat causes their decomposi-
tion. Picrolonic acid is frequently of service in characterizing
bases. Hydrochloric acid precipitates this compound from a
solution of its sodium salt as a yellow, mealy powder, melting
when rapidly heated at about 128, becoming dark in color and
undergoing decomposition with rapid evolution of gas. Knorr
first gave picrolonic acid formula I but formula II 3 is preferred:
I. N0 2 .C 6 H 4 .N II. NO 2 .CeH4.N
I/\.OH N CO
H. Matthes has estimated many alkaloids quantitatively by
1 H. Matthes and O. Rammstedt, Zeitschrift fur analytische Chemie 46, 5^5
(1907) and Archiv der Pharmazie 245, 112 (1907).
2 Berichte der Deutschen chemischen Gesellschaft 30, 914 (1897)-
3 R. Zeine, Inaugural Dissertation, Jena, 1906.
254 DETECTION OF POISONS
means of picrolonic acid. Collect the precipitated alkaloidal
picrolonate in a weighed Gooch crucible, wash, dry and weigh.
Estimation of alkaloids is possible by this method, because the
picrolonates are constant in composition. Morphine, hydras-
tine, codeine, strychnine, brucine, pilocarpine and stypticine 1
can be quantitatively estimated by this method.
Estimation of Morphine, Codeine and Stypticine in Solutions, Tablets
and Sugar Triturations
Dissolve the weighed trituration or tablet in the smallest
quantity of water possible and add picrolonic acid solution
(about o.i n-solution in alcohol) in slight excess. The picro-
lonate separates at once, or very soon, as yellow crystals or a
crystalline meal. Cool 15-30 minutes in ice water and collect
the precipitate in a weighed Gooch crucible. Wash with a
little ice water, dry 30 minutes at 110 and weigh. Morphine
picrolonate usually separates in 10-30 minutes. Cooling aids
precipitation.
Formula Mol. Wt. Decomposition-point
Morphine picrolonate: CnHigNOs.CioHsNAi. 549 200-210
Codeine picrolonate: CisHziNOs.CioEUN^. 563 about 225
Cotarnine picrolonate: C^HisNO^CioHsNAi. 501 205-210
Notes. Practice Analyses: Morphine powder: 0.01-0.02 gram morphine hy-
drochloride, CnHigNOs.HCl.sHkO + 0.5 gram sugar. 0.2-0.5 g ram codeine
phosphate, Cigl^iNOs.HaPO^HzO + 0.5 gram sugar. Stypticine tablets E.
Merck.
Do not use too dilute solutions of the alkaloids in these determinations and do
not wash the picrolonate precipitates with too much water. Dissolve the pow-
dered morphine and sugar mixture in about 5-10 cc. of water. Matthes and
Rammstedt in examining the morphine powder obtained the following results:
Weights taken: 0.019 morphine hydrochloride + 0.5 gram sugar.
Results obtained:
I. 0.0273 -gram morphine picrolonate = 0.0187 gram morphine hydrochloride.
II. 0.02 74 gram morphine picrolonate = 0.0187 gram morphine hydrochloride.
In a second experiment every 10 cc. of an aqueous solution contained 0.0104
gram of morphine hydrochloride.
Results obtained:
I. 0.0147 gram morphine picrolonate = o.oioi gram morphine hydrochloride.
II. 0.0146 gram morphine picrolonate = 0.0099 gram morphine hydro-
chloride. 2
1 Stypticine = cotarnine hydrochloride, Ci2HiBNO 4 .HCl.H 2 O.
2 Professor Matthes has kindly stated that this picrolonate method gives less
satisfactory results with smaller quantities of morphine (0.005 and less;.
SPECIAL QUALITATIVE AND QUANTITATIVE METHODS 255
The application of the picrolonate method to the estimation of hydrastine in
hydrastis root and extract, of nux vomica alkaloids in nux vomica and extract and
of pilocarpine in jaborandum leaves is described in Chapter VI (see pages 282,
296 and 286.
2. Estimation of Alkaloids by Means of Potassium Bismuthous Iodide
(H. Thorns 1 )
Dissolve the particular alkaloid in sulphuric acid and pre-
cipitate completely with potassium bismuthous iodide prepared
as described by Kraut. 2 Decompose the precipitate with a
mixture of sodium carbonate and hydroxide, extract the free
alkaloid with ether and weigh. By this method Thorns has
recovered atropine, hyoscyamine, scopolamine, strychnine,
quinine, caffeine and antipyrine from their potassium
bismuthous iodide precipitates unaltered and nearly quan-
titatively. He has also used this method with success in
estimating quantitatively the alkaloids in belladonna extract.
Procedure. Dissolve the alkaloidal salt, or 2 grams of bella-
donna extract, in 50 cc. of water. Add first locc. of 10 per cent,
sulphuric acid, stir and precipitate with 5 cc. of potassium bis-
muthous iodide solution. Collect the precipitate upon a dry
filter and wash twice with 5 cc. portions of 10 per cent, sulphuric
acid. Transfer the thoroughly drained precipitate and paper
to a wide-mouth extraction cylinder having a tight glass stopper.
Add 0.3 gram of sodium sulphite, then 30 cc. of 15 per cent, so-
dium hydroxide solution and shake. Add quickly 15 grams of
sodium chloride and 100 cc. of ether. Shake frequently and let
stand for 3 hours. The ether contains the alkaloid and settles
well. Remove with a pipette 50 cc. of the ether solution ( =
half the solution of the alkaloidal salt, or i gram of belladonna
extract) and titrate this ether solution in a flask with o.oi
n-hydrochloric acid, using iodeosine as indicator.
After titrating the belladonna alkaloids, use in the calculation
the equivalent weight of atropine-hyoscyamine, Ci7H 23 NO3=
1 Berichte der Deutschen pharmazeutischen Gesellschaft 13, 240 (1903); iS> 85
(1905); 16, 130 (1906) (D. Jonescu).
'Annalen der Chemie und Pharmazie 210, 310 (1882). See "Preparation of
Reagents," page 318.
256 DETECTION OF POISONS
289. 1000 cc. of o.oi n-hydrochloric acid correspond to 2.89
grams of atropine-hyoscyamine. Atropine and hyoscyamine
being isomeric, monacid bases, their formula weight and equiva-
lent weight are the same.
Quinine, caffeine and antipyrine were also recovered un-
altered from potassium bismuthous iodide precipitates. After
decomposition of the precipitates, they were obtained almost
quantitatively but were estimated gravimetrically.
Dissolve 2 grams of quinine 1 in 50 cc. of water acidified with
sulphuric acid and precipitate with potassium bismuthous
iodide. Filter the precipitate with suction and wash with 5
per cent, sulphuric acid. Transfer precipitate and paper to an
extraction cylinder and shake thoroughly with a mixture of 20
grams of crystallized sodium carbonate and 40 cc. of 10 per
cent, sodium hydroxide solution. The yellowish red precipitate
gradually becomes white. Add 30 cc. of ether and shake well
for 30 minutes. Pipette off 25 cc. of the clear ether solution,
evaporate in a weighed glass dish, dry the residue at 100 and
weigh. The weight of quinine was 0.9405 instead of i gram.
Caffeine was estimated in the same way, except that this
alkaloid was extracted with chloroform after decomposition of
the potassium bismuthous iodide precipitate with alkaline
hydroxide and carbonate. The weight of caffeine was 0.9546
instead of i gram.
The precipitate obtained by adding potassium bismuthous
iodide to a solution of antipyrine in sulphuric acid (10 per cent.
H^SO.*) is not decomposed as easily as are those of quinine and
caffeine. The precipitate from 2 grams of antipyrine must be
shaken i hour with 20 grams of sodium carbonate and 60 cc.
of ip per cent, sodium hydroxide solution. Antipyrine must be
extracted with chloroform. The weight of antipyrine was
0.9273 instead of i gram.
Notes. Potassium bismuthous iodide precipitates fixed and volatile alkaloids
but not ammonium salts. If the estimation of volatile bases is unnecessary,
as in the examination of belladonna extract, evaporate the 50 cc. of ether extract
(see above) upon the water-bath. Warm the residue and in a few minutes
1 According to experiments of D Jones cu (loc. ciL}.
SPECIAL QUALITATIVE AND QUANTITATIVE METHODS 257
the strong narcotic odor of volatile bases will disappear. Dissolve the residue
in a little acid-free alcohol and dilute with ether. Before using a flask for
titrations carefully test it beforehand for alkalinity. If a positive test is ob-
tained, alkalinity must be removed. An odor like iodoform, probably due to
the action of sodium hypo-iodite upon the alkaloid, has been observed when sodi-
um hydroxide solution acts upon potassium bismuthous iodide precipitates.
Addition of sodium sulphite may prevent this action. After addition of sodium
chloride ether takes up the alkaloid more readily. But vigorous shaking is
always needed to cause complete transfer of alkaloid to the ether.
3. Estimation of Alkaloids by H. M. Gordin 1
Gordin has found that periodides of the alkaloids, whatever be their composi-
tion, when precipitated from aqueous solution by iodo-potassium iodide in pres-
ence of acids, always contain one equivalent of combined acid for every molecule
of monacid alkaloid. These periodides have the general formula (Alkaloid,
HI) m I n . Iodo-potassium iodide, added to a solution of a monacid alkaloid
acidified with hydrochloric acid, first gives an alkaloid hydrochloride, changed
by potassium iodide to alkaloid hydriodide and finally precipitated as insoluble
periodide by taking up iodine:
(a) Alkaloid + HC1 = Alkaloid.HCl,
(0) Alkaloid.HCl + KI = Alkaloid.HI + KC1,
(7) m (Alkaloid.HI) + In = (Alkaloid.HI) mln = Precipitate.
In the precipitation of an alkaloid in acid solution with iodo-potassium iodide,
one equivalent of acid goes with the precipitate and disappears from solution.
In many cases potassium mercuric iodide may be substituted to advantage for
iodo-potassium iodide. Gordin has found that the composition of the precipi-
tate changes only as regards mercuric iodide and not as far as acid is concerned,
for in this case also the precipitate contains one equivalent of acid for a monacid
alkaloid.
Use in the titration 0.05 n-hydrochloric acid and 0.05 n-potassium hydroxide
solution. Prepare a solution containing a weighed quantity of pure alkaloid, for
example, pure morphine. Dissolve about 0.2 gram of chemically pure morphine,
previously completely dehydrated at 120, in 30 cc. of 0.05 n-hydrochloric acid
in a 100 cc. volumetric flask. Shake and add gradually iodo-potassium iodide
to this solution, until precipitation ceases and the supernatant liquid is dark red.
Dilute to the 100 cc. mark with water and shake vigorously until the liquid above
the precipitate is entirely clear. Pass 50 cc. of solution through a dry filter, de-
colorize the filtrate with a few drops of sodium thiosulphate solution and titrate
excess of 0.05 n-hydrochloric acid with 0.05 n-potassium hydroxide solution, using
phenolphthalein as indicator. Calculate from the result how many grams of
morphine have been neutralized by i cc. of the acid. Comparison of the equiva-
lent weight of morphine with that of any other monacid alkaloid gives the corre-
sponding factor to be used in the calculation. For example, Gordin found in his
experiments that i cc. of approximately 0.05 n-hydrochloric acid neutralized
1 Berichte der Deutschen chemischen Gesellschaft 32, 2871 (1899). Archiv der
Pharmazie 238, 335 (1900). Gordin and A. B. Prescott, Archiv der Pharmazie
237, 380 (1899).
17
258 ' DETECTION OF POISONS
0.0137 gram of anhydrous morphine, CnHigNOs. The factor (x) for strychnine,
C2iH22N 2 O2 (= 334), which is also a monacid base, is as follows:
Morphine : Strychnine
285 : 334 = 0.0137 : x (x = 0.0160)
and that for the monacid base cocaine, Ci7H 21 NO 4 (= 303), according to the
proportion is:
Morphine : Cocaine
285 : 303 = 0.0137 : x (x = 0.0146).
If potassium mercuric iodide is used to precipitate an alkaloid, the method is
the same as with iodo-potassium iodide except that there is no need of treating
the 50 cc. of filtered solution with thiosulphate.
Berberine and colchicin cannot be estimated by Gordin's method.
Quantitative Estimation of Strychnine and Quinine Together
E. F. Harrison and D. Gair 1
Occasionally a small amount of strychnine must be estimated in presence of a
relatively large quantity of quinine, as in certain pharmaceutical preparations. 2
Separation of the two alkaloids is possible by means of Rochelle salt. Quinine
tartrate, (C 2 oH24N 2 O 2 )2.C4H6O 6 .2H 2 O, being difficultly soluble in water, forms
a white crystalline precipitate, whereas strychnine tartrate remains in solution.
Procedure. Render the solution of the mixed alkaloids in about 40 .cc. of
water faintly acid with sulphuric acid. Add enough ammonia to cause a slight
turbidity, then 15 grams of solid Rochelle salt and more ammonia, still leaving
the liquid acid to litmus paper. Heat for 15 minutes upon the water-bath and
then set aside for 2 hours until entirely cold. Filter precipitated quinine tartrate
by suction and wash with aqueous Rochelle salt solution (15 grams of salt in
45 cc. of water) containing 1-2 drops of dilute sulphuric acid. To determine
strychnine, add sodium hydroxide solution to the combined nitrate and wash
water from quinine tartrate until the reaction is alkaline. Extract 2-3 times
with chloroform, pour the chloroform extract through a dry filter and distil in
a weighed flask to about 4 cc. Add 10 cc. of absolute alcohol and evaporate to
dryness upon the water-bath. To remove quinine still adhering to the strych-
nine, extrast the dry residue 2-3 times with i cc. portions of ether, 3 dry at 100
and weigh. This residue consists of pure, quinine-free strychnine.
Estimation of Toxicity of Chemical Compounds by Blood Haemolysis
(A. J. J. Vandevelde 4 )
Vandevelde originally used living cells of a variety of Allium cepa (red Bruns-
wick onion), the cell membrane of which is rich in anthocyan. The presence of
this substance obviated the necessity of using a special coloring matter in
determining plasmolytically the toxicity of alcohols, ethereal oils and other
substances. 6 Vandevelde has recently recommended determining the toxicity of
chemical compounds by blood haemolysis, using for this purpose defibrinated ox
1 Pharmaz. Journ. (4) 17, 165.
2 Compound Syrup of Hypophosphites, U. S. P.
3 More ether dissolves a weighable quantity of strychnine.
4 Chemiker Zeitung 29, 565 (1905).
6 Bulletin de 1' Association Belgee de Chimie 17, 253.
SPECIAL QUALITATIVE AND QUANTITATIVE METHODS 259
blood (see pages 229 and 230). To establish the toxicity of different alcohols, the
concentration at which haemolysis just ceases is determined. A solution, in which
blood-corpuscles are not hydrolyzed after a definite time but are hydrolyzed
upon addition of the slightest trace of the substance being examined, is a non-
toxic solution for blood-corpuscles, called by Vandevelde a "critical solution."
The estimation requires:
1. A solution of 0.9 per cent, sodium chloride in 50 per cent, alcohol by volume. 1
2. An aqueous 0.9 per cent, sodium chloride solution.
3. A suspension of 5 per cent, defibrinated ox blood in 0.9 per cent, aqueous
sodium chloride solution.
Experiments are made in test-tubes. Place first in each of several tubes 2.5 cc.
of the mixtures (of different concentration) of alcoholic and aqueous sodium
chloride solution and then 2.5 cc. of the suspended blood. The end-point for
the appearance of haemolysis was set at three hours.
Vandevelde's experiments with ethyl alcohol gave the following results:
Alcoholic con-
Cc. of alcoholic Cc. of aqueous
centration of
After 3
Cc. of sus-
NaCl solu-
NaCl solution
mixture in vol.
hours
pended blood
tion
per cent.
2-5
2.20
0.30
22. O
Haemolysis
5
2-15
0-35
21-5
Haemolysis
5
2. 10
0.40
21.0
Haemolysis
5
2.05
o.4S
20.5
Haemolysis
5
2.00
0.50
2O. O
Haemolysis
5
i-9S
o-SS
19-5
No haemolysis
5
1.90
0.60
19.0
No haemolysis
Consequently the critical solution of ethyl alcohol is one containing 19.5 cc. of
absolute alcohol (C 2 HO) in 100 cc., or 15.489 grams of C 2 H 6 O in 100 cc. The
specific gravity of absolute alcohol being 0.7943, 19.5 cc. weigh 19.5 X 0.7943
15-489.
According to Vandevelde's experiments addition of methyl alcohol diminished
the toxicity of ethyl alcohol, whereas the higher alcohols were found to be more
toxic than the latter. If the toxicity of 100 parts by weight of ethyl alcohol is
taken as 100, then 47 parts by weight of isopropyl, 29 parts of isobutyl and 12.5
parts of amyl alcohol are isotoxic with that quantity of ethyl alcohol. Using
the plasmolytic method with onion cells, Vandevelde obtained the following
results with the same series:
100, 36.8, 21.2, 12.6.
The hzemolytic method is easily performed in test-tubes and does not require
the use of the microscope. The form of the tube, especially its diameter, is quite
important in these experiments. The speed of haemolysis increases with the
diameter of the tube. The quantity of the blood-corpuscles is of slight influence
except in narrow tubes and at the beginning of the reaction. Vandevelde applies
the -term "critical coefficient" to the number giving the concentration of a sub-
stance necessary to kill the cells.
1 The specific gravity of such an alcohol at 15 is 0.9348.
CHAPTER VI
QUANTITATIVE ESTIMATION OF ALKALOIDS AND OTHER
PRINCIPLES
Estimation of Alkaloids in Drugs and Pharmaceutical Preparations
(German Pharmacopoeia)
Alkaloids are nitrogenous bases occurring in plants. The
term "plant base" is synonymous with alkaloid. The plant
families especially rich in alkaloids are berberideae, cinchonaceae,
papaveracese, solanaceae and strychnaceae. Alkaloids as a rule
are not uniformly distributed in all parts of plants. They
occur most often in roots, fruits and seeds. If the plant is a
tree, there is often more alkaloid in the bark than in other parts.
The particular part of the plant usually contains only a few per
cent, of alkaloid. Quinine bark is an exception, the quantity
of alkaloid being 5-10 per cent, and sometimes more. Plants as
a rule do not contain free alkaloids but their salts. They are
combined not only with the mineral acids, sulphuric, hydro-
chloric and possibly phosphoric, but with organic acids, as malic,
aconitic, tannic, citric, quinic and meconic. Most free plant
bases dissolve only slightly in water but readily in ether and
chloroform. The German Pharmacopoeia prescribes a mixture
of ether and chloroform for the extraction of free alkaloids.
The finely powdered drug should first be treated with a solution
of sodium hydroxide, ammonia or sodium carbonate to liberate
alkaloids from their salts:
C 20 H 24 N 2 O 2
C 6 H 7 (OH) 4 COOH + NaOH = C 20 H 24 N O 2 + C 6 H 7 (OH)4COONa + HzO.
Quinine quinate 1 Quinine Sodium quinate
The ether-chloroform mixture removes not only alkaloids
from the drug but varying amounts of other substances, as fat,
1 Cinchona bark contains quinine in the form of this salt.
260
QUANTITATIVE ESTIMATION OF ALKALOIDS 261
resin, wax and pigments. To free the alkaloid from such im-
purities, shake the ether-chloroform extract with a measured
excess of o.i or o.oi n-hydrochloric acid. The alkaloid passes
into aqueous solution as hydrochloride :
C 2 oH 24 N 2 2 + HC1 = C 2 pH 24 N 2 2 .HCl
Quinine Quinine hydrochloride
But the impurities remain in the ether-chloroform mixture.
Finally, determine excess of hydrochloric acid by titration with
o.i or o.oi n-potassium hydroxide solution, employing usually
iodeosine as indicator. Calculate the amount of alkaloid in the
drug from the difference between the original quantity of acid
and the excess.
The estimation of alkaloids in drugs and pharmaceutical
preparations, according to directions given by the German
Pharmacopoeia, requires the following steps:
1. Liberation of alkaloids from salts by means of stronger
bases, as potassium and sodium hydroxides, ammonia and
sodium carbonate.
2. Extraction of free alkaloids with ether-chloroform mixture.
3. Transference of alkaloids from ether-chloroform to aque-
ous o.i or o.oi n-hydrochloric acid solution.
4. Determination of excess of hydrochloric acid in an aliquot
volume, usually 50 cc. of the hydrochloric acid solution of the
alkaloid diluted to 100 cc., by titration with o.i or o.oi n-potas-
sium hydroxide solution.
Alkaloids in Aconite Root
Officinal aconite root is the root of Aconitum Napellus col-
lected at the end of flowering. Two alkaloids are present,
namely, aconitine, Cs^rNOn, and picraconitine, C 3 iH 4 7NOio
(?), characterized by its very bitter taste. Both alkaloids are
combined with aconitic acid,
CH.COOH
C.COOH
CH 2! COOH
262 DETECTION OF POISONS
Boiled with water, or alcoholic potassium hydroxide solution,
aconitine yields a new base, aconine, benzoic and acetic acids: 1
On + 2 H 2 O = CzsH^NOs + C H 4 O 2 + C 6 H 6 .COOH.
Aconitine Aconine Acetic Benzoic acid
acid
This reaction presents aconitine as acetyl-benzoyl-aconine,
/COCHa
C 26 H 3 9NO9<
X COCH 6 .
Since aconitine has been shown to contain four methoxyl
groups, the formula of this alkaloid may be written:
/COCH,
C 2 iH 27 (OCH 3 )4N0 6 <
\COC 6 H 6 .
Aconine, therefore, is CaiHarCOCHsMOH^NOs, and picra-
conitine must be regarded as benzoyl-aconine, having the for-
mula C 2 iH 21 (OCH 3 ) 4 (OH)NO4(COC6H 5 ).
Estimation of Aconitine
(German Pharmacopoeia)
Place 12 grams of rather finely powdered aconite root dried at 100 in an
Erlenmeyer flask and add 90 grams of ether and 30 grams of chloroform. Shake
well and add 10 cc. of a mixture of 2 parts of sodium hydroxide solution and i
part of water. Let the mixture stand 3 hours, shaking vigorously at frequent
intervals. Then add 10 cc. of water, or enough to cause the powder to gather
into balls after vigorous shaking and leave the supernatant ether-chloroform
solution perfectly clear. After an hour pass 100 grams of the clear ether-
chloroform solution through a dry filter kept well covered and receive the filtrate
in a small flask. Distil about half the solvent and pour the remainder into a
separating funnel. Wash the flask with three 5 cc. portions of a mixture of 3
parts of ether and i part of chloroform, and thoroughly shake the combined
solutions with 25 cc. of o.oi n-hydrochloric acid. When the liquids have
separated perfectly clear, add enough ether to bring the ether-chloroform solu-
tion to the surface. Pass the acid solution through a small filter moistened with
water and collect the filtrate in a 100 cc. flask. Make three more extractions
of the ether-chloroform solution with 10 cc. portions of water, and pass these
extracts through the same filter. Wash the latter with water and dilute the total
solution with water to TOO cc. Place 50 cc. of this solution in a 200 cc. flask with
50 cc of water and enough ether to make a layer about i cm. thick. Add 5
drops of iodeosine solution and run in, while shaking, enough o.oi n-potassium
hydroxide solution to turn the aqueous solution pale red. '
x Freund and Beck, Berichte der Deutschen chemischen Gesellschaft 27,
433. 720 (1894); 28, 192, 2537 (1895).
QUANTITATIVE ESTIMATION OF ALKALOIDS 263
Calculation. Dissolve the aconite alkaloids set free from their salts by
sodium hydroxide solution in 120 grams of ether-chloroform. Weigh 100 grams
of this solution (= alkaloids from 10 grams of aconite root). Dissolve the
alkaloids with 25 cc. of o.oi n-hydrochloric acid, bringing the volume to roo cc.
Determine excess of acid in 50 cc. of this solution (= alkaloids from 5 grams of
root). If, for example, this requires 8.5 cc. of o.oi n-potassium hydroxide
solution, then the alkaloids have combined with 12.5 - 8.5 =4 cc. of o.oi n-acid.
Since the equivalent weight of aconitine CsJ^NOu = 645, 100 cc. of o.oi
n-hydrochloric acid unite with 6.45 grams of aconitine. The proportion
1000 : 6.45 = 4 : x (x = 0.0258)
shows that 5 grams of aconite root contain 0.0258 gram o* alkaloids, correspond-
ing to 0.51 per cent. The German Pharmacopoeia demands this quantity of
aconitine in aconite root as a minimum. Using a different method, the United
States Pharmacopoeia has the same limit.
Estimation of Cantharidin in Spanish Flies
(German Pharmacopoeia)
Place 25 grams of Spanish flies ground mediumly fine in an Erlenmeyer flask
and add 100 grams of chloroform and 2 cc. of hydrochloric acid. 1 Shake the
mixture frequently during 24 hours. Then pour 52 grams of the chloroform solu-
tion through a dry filter kept well covered, and collect the nitrate in a weighed
flask holding 80-100 cc. Distil the chloroform, and add 5 cc. of petroleum ether
to the residue. Stopper the flask, and let the mixture stand 12 hours, with oc-
casional agitation. Dry at 100 and weigh a filter (5 cm. in diameter). Pass
the liquid through this filter, having first moistened it with petroleum ether.
Treat the undissolved residue twice with petroleum ether, each time using 10 cc.
and shaking. Pass this solvent through the same filter, and disregard crystals
adhering to the side of the flask. Dry the filter and flask, and wash both with
a little water, containing a drop of ammonium carbonate solution to every 10 cc.,
until this solvent is only faintly yellow. Finally, wash once with 5 cc. of water,
and dry both flask and filter. Place filter and contents in the flask, and dry at
100 to constant weight. The crystalline residue should weigh at least o.i gram.
Notes. Additional information about cantharidin is given
on page 203. Spanish flies contain cantharidin partly free and
partly as an alkali salt of cantharidic acid (cantharidate) .
Hydrochloric acid sets cantharidic acid free and the latter then
passes at once into cantharidin, its internal anhydride. Conse-
quently hydrochloric acid is essential to the determination
of that cantharidin present in Spanish flies as cantharidate.
Chloroform not only dissolves cantharidin but fatty_substances
1 Specific gravity 1.124 = 2 S P er cent. HC1.
264 DETECTION OF POISONS
in the flies. To isolate pure cantharidin from these impurities,
distil the chloroform and let the residue stand for 1 2 hours in the
cold with petroleum benzene. Fat readily dissolves but can-
tharidin is as good as insoluble in this solvent. The German
Pharmacopoeia finally directs weighing the cantharidin from
12.5 grams of powdered Spanish flies. The quantity should be
at least o.i gram, corresponding to 0.8 per cent, of cantharidin
as a minimum. With sufficient care, white crystalline can-
tharidin may be isolated from Spanish flies.
Baudin obtained from good flies 1.06 per cent, of cantharidin,
of which 0.72 per cent, was free and 0.34 per cent, combined
as cantharidate. Dieterich found only 0.3 per cent, of free
cantharidin.
Estimation of Cinchona Alkaloids
(German Pharmacopoeia)
i. In Cinchona Bark. To determine total alkaloids, pour 90 grams of ether
and 30 grams of chloroform upon 12 grams of finely ground cinchona bark,
dried at 100 and placed in an Erlenmeyer flask. Add 10 cc. of sodium hydroxide
solution. Shake vigorously at frequent intervals during 3 hours. Then add 10
cc. of water, or enough to cause the powdered cinchona to gather into lumps
after vigorous shaking, thus leaving the supernatant ether-chloroform solution
perfectly clear. Let the ether-chloroform solution ?tand an hour, and then pass
100 grams through a dry filter, kept well covered. Collect the filtrate in a flask,
and distil half the solvent. Pour the remaining ether-chloroform solution into
a separating funnel, and wash the flask three times with 5 cc. portions of a
mixture of 3 parts of ether and i part of chloroform. Thoroughly extract the
total ether-chloroform solution with 25 cc. of o.i n-hydrochloric acid. When the
contents of the separating funnel are perfectly clear, add enough ether to bring
the ether-chloroform solution to the surface. Pass the acid solution through a
small filter moistened with water, and receive the filtrate in a 100 cc. flask.
Make three more extractions of the ether-chloroform solution with 10 cc. portions
of water, and pass these extracts through the same filter. Wash the filter with
water, and bring the volume of the filtrate to 100 cc. Finally, measure 50 cc.
of this solution with a pipette, and add freshly prepared hsematoxylm solution,
made by dissolving a small particle of this substance in i cc. of alcohol. Shake
and add enough o.i n-potassium hydroxide solution to give the mixture a yel-
lowish color, which quickly changes after vigorous agitation to bluish violet. 1
Notes and Calculation. Both quinine and quinidine have
the formula C 20 H 2 4N 2 O2 and cinchonine and cinchonidine the
x The German Pharmacopoeia prescribes that not more than 4.3 cc. of o.i
n-potassium hydroxide should be required.
QUANTITATIVE ESTIMATION OF ALKALOIDS 265
formula Ci 9 H 22 N 2 O. These are the most important alkaloids
in cinchona bark. They are present in all true cinchona barks
as salts of quinic acid, CnHiaOe, and quino-tannic acid. Fuller
information regarding the chemistry of quinine and cinchonine
is given on page 119.
Quinic acid is widespread in the vegetable kingdom. This
monobasic, pentatomic acid, having the formula, C 6 H 7 (OH) 4 .
COOH, is a hexahydro-tetroxy-benzoic acid. It crystallizes
in large monoclinic prisms melting at 162. As far as the
chemical behavior of quinic acid is concerned, either of the
following formulas is possible:
I. H OH II. H OH
H 2 C CH.OH H 2 C CH.OH
H 2 C CH.OH HO.HC CH 2
X X
HO COOH HO COOH
The formation of tetra-acetyl-quinic acid,
C 6 H 7 COOH, and tetra-benzoyl-quinic acid,
COOH, shows that quinic acid contains four alcoholic hydroxyl
groups. Addition of sodium hydroxide solution to cinchona
powder sets the alkaloids free from their salts:
C 6 H 7 (OH) 4 COOH + NaOH = C 20 H 24 N 2 O 4 + H 2 O + C 6 H 7 (OH)4COONa.
Quinine quinate Quinine Sodium qumate
Only 100 grams of the original 120 grams of ether-chloroform
mixture (=12 grams of cinchona powder) are in the nitrate.
This solution contains the alkaloids in 10 grams of bark.
These 100 grams are extracted with 25 cc. of o.i n-hydrochloric
acid, the alkaloids passing into aqueous solution as hydro-
chlorides, and the volume is brought to 100 cc. Finally,
excess of a.i n-hydrochloric acid in 50 cc. (= alkaloids in 5
grams of bark) of this hydrochloric acid solution is determined
by titration. In these determinations with very dilute hydro-
266 DETECTION OF POISONS
chloric acid, cinchona alkaloids behave as monacid bases, 1
quinine forming C 2 oH 24 N 2 O 2 .HCl and cinchonine Ci9H 22 N 2 O.-
HC1.
The mean of the equivalent weight of quinine (324) and
cinchonine (294), that is to say, (324 + 294) divided by 2 = 309,
may be taken as the equivalent weight. This value agrees
approximately with the actual quantities of these alkaloids
in cinchona bark. Consequently 1000 cc. of o.i n-hydrochloric
acid are equivalent to 30.9 grams of cinchona alkaloids.
Example. Titration of 50 cc. of the hydrochloric acid solution of alkaloids, in
preparing which 12.5 cc. of o.i n-hydrochloric acid were used, required 2.6 cc.
of o.i n-potassium hydroxide solution, equivalent to the volume of o.i n-hydro-
chloric acid in excess. 12.5-2.6 = 9.9 cc. of o.i n-hydrochloric acid have
combined with the alkaloids in 5 grams of cinchona bark. The proportion
Cc. o.i n-HCl:Grams of Alkaloids
1000 : 30.9 = 9.9 : x (x = 0.30591)
shows that 5 grams of bark contain 0.30591 gram of alkaloids. Consequently
100 grams of bark contain 20 X 0.30591 = 6.11 grams of alkaloids.
Titrate the filtered ether-chloroform solution of cinchona alkaloids at once.
The solution should not be exposed for any length of time to direct sunlight.
Otherwise chloroform may give free hydrochloric acid
CHC1 3 + O = COC1 2 + HC1
which will neutralize alkaloids. The decomposition of 0.05 gram of chloro-
form would give enough hydrochloric acid to neutralize 0.25 gram of cinchona
alkaloids. Panchaud 2 has shown that such chloroform solutions of cinchona
alkaloids after standing 12 hours yield only 80 per cent, of the total quantity of
alkaloids originally present.
Haematoxylin, CieHuOe-sHzO, occurs in logwood, the heart-wood of Haema-
toxylon campechianum. It usually crystallizes in colorless, shining, quadratic
prisms containing 3 molecules of water, more rarely in rhombic crystals with i
molecule of water. It dissolves only slightly in cold water but freely in boiling
water, alcohol or ether. In contact with air haematoxylin gradually becomes
reddish.
2. In Aqueous and Alcoholic Cinchona Extracts. To determine total alkaloids
in these preparations, dissolve 2 grams of the given extract in an Erlenmeyer
flask, using 5 grams of water and 5 grams of absolute alcohol. Add 50 grams of
1 Quinine dihydrochloride, C2oH 2 4N2O2.2HCl, is formed by passing gaseous
hydrogen chloride over quinine and also by dissolving the monohyflrochloride,
C 2 oH24N 2 O2.HCl, in strong hydrochloric acid with gentle heat. An aqueous
solution of the dihydrochloride has an acid reaction.
2 Schweizer Wochenschrift fur Pharmazie 44, 580.
QUANTITATIVE ESTIMATION OF ALKALOIDS 267
ether and 20 grams of chloroform, and, after vigorous shaking, 10 cc. of sodium
carbonate solution (1:3). Shake frequently, and let the mixture stand an hour.
Then pass 50 grams of the ether-chloroform solution through a dry filter, kept
well covered. Receive the filtrate in a flask, and distil half the solvent. Pour
the remainder into a separating funnel, wash the flask three times with 5 cc.
portions of a mixture of 3 parts of ether and i part of chloroform, and then shake
the total ether-chloroform solution with 10 cc. of o.i n-hydrochloric acid. When
the contents of the separating funnel are perfectly clear, add enough ether to bring
the ether-chloroform solution to the surface. Pass the acid solution through a
small filter moistened with water, and receive the filtrate in a 100 cc. flask. Make
three more extractions of the ether-chloroform solution with 10 cc. portions of
water, and pass these extracts through the same filter. Wash the filter with
water and bring the volume of the filtrate to 100 cc. Finally, measure 50 cc. of
this solution with a pipette, and add freshly prepared haematoxylin solution,
made by dissolving a small particle of this substance in i cc. of alcohol. Shake
and add enough o.i n-potassium hydroxide solution to give the mixture a yellowish
color, which quickly changes upon vigorous agitation to bluish violet.
Notes and Calculation. The alkaloids in the two cinchona
extracts are set free from their salts by sodium carbonate:
2 | + Na 2 CO 3 = 2C 20 H 2 4N 2 O 2 + 2C 6 H 7 (OH) 4 COONa + H 2 O + CO 2 .
C 6 H 7 (OH) 4 COOH
Quinine Quinine Sodium
quinate quinate
The alkaloids from 2 grams of extract are dissolved in 75
grams of alcohol-ether-chloroform mixture. Two-thirds of this
solution, or 50 grams (= alkaloids in 1.33 grams of extract)
are used in the determination. The free alkaloids in this por-
tion pass into aqueous solution as hydrochloride, C 20 H 24 N 2 O 2 .-
HC1, upon extraction with 10 cc. of o.i n-hydrochloric acid.
The excess of hydrochloric acid in half of this solution diluted
to 100 cc., that is to say, in 50 cc. (= alkaloids in 0.666 gram
of extract), is finally determined by titration. If 3.7 cc. of
o.i n-potassium hydroxide solution are required, 5 3-7 =
1.3 cc. of o.i n-hydrochloric acid have combined with the
alkaloids in 0.666 gram of cinchona extract. The mean
equivalent weight of quinine and cinchonine (= 309), used in
the proportion:
1000 130.9 = 1.3 :x (x = 0.04017)
shows that 1.3 cc. of o.i n-hydrochloric acid correspond to
0.04017 gram of alkaloids, or 6.03 per cent. The German
268 DETECTION OF POISONS
Pharmacopoeia demands this quantity as a minimum for the
aqueous extract of cinchona bark.
In the determination of alkaloids in alcoholic cinchona ex-
tract, titration of excess of o.i n-hydrochloric acid in 50 cc.
should not require more than 2.3 cc. of o.i n-potassium hy-
droxide solution. Then 5 2.3 = 2.7 cc. of o.i n-hydrochloric
acid represent the alkaloids in this solution. This extract at
the minimum must contain 12.55 P er cent - of alkaloids.
Sulphate Method of Estimating Quinine in Mixtures of Cinchona
Alkaloids
(J. Carles) 1
This method is especially recommended for practical purposes
because of its accuracy and simplicity. Differences in the
solubilities of the sulphates of cinchona alkaloids in ammonium
sulphate solutions form the basis of the method. E. Schmidt
has found that these sulphates have the following solubilities
in water at 15:
Quinine sulphate i : 800 Cinchonine sulphate i : 65
Quinidine sulphate i : 100 Cinchonidine sulphate i : 97.
Guareschi has found quinine sulphate practically insoluble
in an ammonium sulphate solution, a result which Hille 2 has
confirmed. An addition of 0.0078 gram to the quantity of
quinine sulphate obtained is necessary on account of the
quinine sulphate in the 20 cc. of wash water used.
i. Cinchona Bark. Place 12 grams of finely powdered cinchona bark dried at
100 in an Erlenmeyer flask and add 90 grams of ether and 30 grams of chloro-
form. Add 10 cc. of sodium hydroxide solution. Shake vigorously at frequent
intervals for 3 hours. Then add 10 cc. of water, or enough to cause the powdered
cinchona to gather into balls after thorough shaking and leave the supernatant
ether-chloroform layer perfectly clear. After i hour pass 100 grams of the
ether-chloroform solution through a dry filter kept well covered. Collect the
nitrate in a dry weighed flask, distil the ether-chloroform and dry the flask at 1 10
to constant weight. The increase in the weight of the flask corresponds to the
total alkaloids in 10 grams of bark.
1 Zeitschrift fur analytische Chemie 9, 467 (1870).
2 W. Hille (Archiv der Pharmazie 241, 54 (1903), has reviewed critically the
various methods that have appeared thus far for the estimation of quinine in pres-
ence of other cinchona alkaloids.
QUANTITATIVE ESTIMATION OF ALKALOIDS 269
Warm the alkaloidal residue in the flask with water and dilute sulphuric acid
and niter the solution. Wash the flask 3 times with water containing sulphuric
acid and pour the wash water through the same filter. Dilute the nitrate to
about 50 cc., heat to boiling and exactly neutralize with ammonia. Cool and
after 6 hours collect upon a weighed filter, or better in a Gooch crucible, the
flocculent precipitate of quinine sulphate, wash with 20 cc. of cold water, dry at
110 and weigh.
Add 0.0078 gram to the weight of quinine sulphate found and calculate the
quantity of quinine in 10 grams of cinchona bark as follows:
C2 H24N 2 O2.H 2 SO4:C2oH24X 2 O2 = Quinine sulphate + 0.0078 :x.
(746) (648) found
2. Cinchona Extract. Dissolve 3 grams of aqueous cinchona extract in 5 grams
each of water and absolute alcohol and place in a measuring cylinder. Add 50 cc.
of ether, 10 cc. of chloroform and, after shaking vigorously, 10 cc. of sodium car-
bonate solution (1:3). Shake at frequent intervals during 3 hours. When two
layers have formed, bring the ether layer to the 75 cc. mark with more ether.
Rotate the container carefully and evaporate 50 cc. of the clear ether-chloroform
layer in a dry weighed flask. Dry i hour at 105 and weigh when cold. The in-
crease in weight corresponds to the total alkaloids in 2 grams of cinchona extract.
There should be at least 0.12 gram, or 6 per cent, of alkaloid.
To determine quinine, pour very dilute sulphuric acid over the weighed alka-
loidal residue in the flask, warm and filter. Rinse the flask several times with
very dilute sulphuric acid, bring the volume to about 50 cc. with water and pro-
ceed as directed above under cinchona bark. Collect the quinine sulphate in 2
hours upon a weighed filter, or Gooch crucible. The calculation is the same as
for cinchona bark.
Estimation of Colchicin in Colchicum Seed and Conns
(J. Katz and G. Bredemann 1 )
Exhaust colchicum seed or corms with 60 per cent, alcohol
and evaporate 50 grams of this extract to 20 cc. Add 0.5
gram of solid paraffine and 20 cc. of water. Warm until the
paraffine is melted and the alcohol has been completely expelled.
Cool the liquid evaporated to 10-15 cc - anc * P ass through a
moist filter. Melt the paraffine cake upon the water-bath with
10 cc. of 10 per cent, acetic acid and pour the cold liquid through
the same filter. Wash the latter, the paraffine cake and the
dish with water. Saturate the total filtrate with sodium chlo-
ride and extract first with 20 cc. of chloroform and then with
10 cc. portions until a few drops of the aqueous liquid show
.scarcely any turbidity with 0.05 n-iodine solution. Pass the
chloroform solution through a filter moistened with this solvent
1 Pharmazeutsche Zentral-Halle 42, 289 and Apotheker-Zeitung 18, 817-
270 DETECTION OF POISONS
and evaporate. To expel chloroform retained by the colchicin,
dissolve the residue in a little water and filter. Evaporate the
solution in a weighed dish and dry the residue over sulphuric
acid to constant weight.
Note. Using this method, Bredemann obtained the following quantities of
colchicin:
In seed 0.46 -0.13 per cent.
In conns 0.032-0.06 per cent.
In fresh flowers 0.6 per cent.
In dry flowers 1.8 per cent.
Alkaloids in Pomegranate Bark
Pomegranate bark, the bark of Punka Granatum, contains
the following four alkaloids :
Pelletierine, C 8 H 15 NO, Methyl-pelletierine, C 9 H 17 NO,
Isopelletierine, C 8 H 15 NO, Pseudo-pelletierine, C 9 H 15 NO.
According to Piccini there is still another alkaloid in the bark of
pomegranate root isomeric with methyl-pelletierine and there-
fore called isomethyl-pelletierine.
Ciamician and Silber have determined the structure of
pseudo-pelletierine which they call n-methyl-granatonine.
Pseudo-pelletierine (I) is a ketone which, upon treatment with
sodium amalgam or with sodium and alcohol, adds two atoms
of hydrogen and passes into the corresponding secondary
alcohol, n-methyl-granatoline (II). Chromic and sulphuric
acids oxidize the latter to n-methyl-granatic acid (III) . Nitro-
gen can be eliminated by exhaustive methylation and the final
product is normal suberic acid (IV) :
I. H 2 C CH CH 2 II. H 2 C CH CH 2
H 2 C N.CH 3 CO H 2 C N.CHsCH.OH
H 2 C CH CH 2 ' H 2 C CH CH 2
Pseudo-pelletierine = n-methyl-granatolin
n-methyl-granatonine
III. H 2 C CH COOH IV. H 2 C CH 2 .COOH
H 2 C N.CH 3 > H 2 C
H 2 C CH CH 2 CO.OH H 2 C CH 2 .CH 2 .COOH
n-methyl-granatic acid Normal suberic acid
QUANTITATIVE ESTIMATION OF ALKALOIDS 271
Estimation of Alkaloid in Pomegranate Bark
(German Pharmacopoeia)
To determine total alkaloids, pour 90 grams of ether and 30 grams of chloro-
form upon 12 grams of rather finely ground pomegranate bark, dried at 100 and
placed in an Erlenmeyer flask. Shake vigorously and add 10 cc. of a mixture of
2 parts of sodium hydroxide solution and i part of water. Let the mixture stand
3 hours, shaking vigorously at frequent intervals. Then add 10 cc. of water,
which will cause the powder to gather into balls after vigorous shaking, and leave
the supernatant ether-chloroform solution perfectly clear. After an hour, pass
100 grams of the clear ether-chloroform solution through a dry filter, kept well
covered and receive the filtrate in a separating funnel. Extract this filtrate
with 50 cc. of o.oi n-hydrochloric acid and pass this acid solution, when perfectly
clear, through a small filter moistened with water into a TOO cc. flask. Make
three more extractions with 10 cc. portions of water, and pass these extracts
through the same filter. Wash the filter with water and dilute the total solution
with water to 100 cc. Place 50 cc. of this solution in a 200 cc. flask with 50 cc.
of water and enough ether to make a layer about i cm. thick. Add 5 drops of
iodeosine solution and enough o.oi n-potassium hydroxide solution, shaking
vigorously after each addition, to give a pale red color to the lower aqueous
solution.
Calculation. The 100 grams of filtered ether-chloroform solution correspond
to 10 grams of bark. The alkaloids are transferred from this ether-chloroform
solution to 50 cc. of o.oi n-hydrochloric acid which are diluted with water to
100 cc. The excess of hydrochloric acid in 50 cc. of this solution (= alkaloids
from 5 grams of bark) is determined by titration. ^If, for example, n cc. of o.oi
n-potassium hydroxide solution are used, then 25 n = 14 cc. of o.oi n-hydro-
chloric acid have combined with the alkaloids in 50 cc. of the solution. If the
mean of the equivalent weights of pelletierine (141) and pseudo-pelletierine
(153), or 147, is used in the calculation, 1000 cc. of o.oi n-hydrochloric acid neu-
tralize 1.47 grams of the mixed alkaloids. According to the proportion
1000 : 1.47 = 14 : x (x = 0.02058)
5 grams of pomegranate bark contain 0.02058 gram of alkaloids which corre-
sponds to an alkaloid content of 20 X 0.02058, or 0.41 per cent. The German
Pharmacopoeia demands this quantity of alkaloids in pomegranate bark as a
Estimation of Caffeine in Coffee, Tea, Cola Nuts and Guarana
(Literature)
A. Hilger and A. Juckenack. Zur Bestimmung des Kaffeins in Kaffee und
Tee. Forschungsberichte iiber Lebensmittel und ihre Beziehungen zur Hy-
giene 4, 40-50; C 1 1897 I, 775 and also 4, I45~ I S4 and C 1897 II, 233.
H. Trillich and H. Gockel . Beitrage zur Kenntniss des Kaffees und der
Kaffeesurrogate. Forschungsberichte iiber Lebensmittel und ihre Beziehungen
zur Hygiene 4, 78-88 and C 1897 I, 1248.
1 C = Chemisches Zentralblatt.
272 DETECTION OF POISONS
L. Graf. Ueber Zusammenhang von Kaffemgehalt und Qualitat bei chines-
ischen Tee. Forschungsberichte ueber Lebensmittel und ihre Beziehungen zur
Hygiene 4, 88-89, and C 1897 I, 1249.
A. Forster and R. Riechelmann. Zur Bestimmung des Kaffeins im Kaffee.
Zeitschrift fur 6'ffentliche Chemie 3, 129-131 and C 1897 I, 1259.
C. C. Keller! Die Bestimmung des Kaffeins im Tee. Berichte der Deutschen
pharmaceutischen Gesellschaft 7, 105-112 (1897) and C 1897 I, 1134.
A. Forster and A. Riechelmann. Zur Bestimmung des Kaffeins im Kaffee.
(Entgegnung.) Zeitschrift fiir offentliche Chemie 3, 235-236 and C 1897 II,
436.
E. Tassily. Ueber ein neues Verfahren zur Bestimmung des Kaffeins im
Kaffee. Bulletin de la Societe" chimique, Paris, (3) 17, 766-768 and C 1897 11,
644.
K. Dieterich. Ueber die Werthbestimmung der Kolanuss und des Kolaex-
traktes. Vortrag auf der Naturforscherversammlung in Braunschweig gehalten.
Pharmaceutische Zeitung 42, 647-650 and C 1897 II, 977.
H. Brunner and H. Leins. Ueber die Trennung und quantitative Bestim-
mung des Kaffeins und Theobromins. Schweizer Wochenschrift fiir Pharmacie
36, 301-303 and C 1898 11, 512.
J. Gadamer. Ueber Kaffeinbestimmungen in Tee, Kaffee und Kola. Archiv
der Pharmacie 237, 58-68 and C 1899 I, 713.
E. Katz. Ueber die quantitative Bestimmung des Kaffeins. Berichte der
Deutschen pharmaceutischen Gesellschaft 12, 250 (1902).
i. C. C. Keller's Method. Pour 120 grams of chloroform
upon 6 grams of dry, unbroken tea leaves 1 in a wide-mouth
separating funnel. In a few minutes, add 6 cc. of ammonium
hydroxide solution (10 per cent. HaN), and at frequent intervals
shake vigorously during 30 minutes. Then let the separating
funnel stand at rest, until the solution is perfectly clear and the
tea leaves have absorbed all the water. This may require
3-6 hours, or even longer, depending upon the variety of tea.
Pass 100 grams of clear chloroform extract, representing 5
grams of tea, through a small filter moistened with chloroform.
Receive the filtrate in a small, weighed flask and distil the chloro-
form upon the water-bath. Pour 3-4 cc. of absolute alcohol
over the residue. Heat upon the water-bath to remove alcohol
and expel alcohol vapor with a hand bellows. In a few minutes
1 When a wide-mouth separating funnel cannot be obtained, triturate the
tea leaves somewhat, solely to facilitate their removal from the separating funnel
after extraction. Finely powdered tea is not only unnecessary, but even ob-
jectionable, because the extracts have a much deeper color, and the yield of
caffeine is not increased.
QUANTITATIVE ESTIMATION OF ALKALOIDS 273
the caffeine will be dry and at the same time free from im-
prisoned chloroform. In a measure also, this treatment with
alcohol separates caffeine from extraneous chlorophyll. The
lattei adheres to the bottom and side of the flask, whereas
caffeine forms a white incrustation upon it. Caffeine thus
obtained is usually impure from small quantities of ethereal
oil, fat, vegetable-wax and principally chlorophyll. Conse-
quently, it must be purified. Set the flask upon a boiling water-
bath and pour a mixture of 7 cc. of water and 3 cc. of alcohol
over the crude caffeine, which upon being shaken will pass into
solution almost immediately. Then add 20 cc. of water,
stopper the flask and shake vigorously. The chlorophyll will
form lumps and the solution will filter easily. Pass the caffeine
solution through a small filter moistened with water, wash flask
and filter with 10 cc. of water, evaporate the total filtrate in
a weighed glass dish to dryness upon the water-bath and weigh
the residue of nearly pure caffeine. The weight of this residue
multiplied by 20 will give the percentage of caffeine in the tea.
Notes. Ammonia causes tea leaves to swell considerably,
and at the same time combines with the tannic acid present.
Caffeine is set free and dissolved by chloroform. The color of
the chloroform extract depends upon the variety of tea. Black
teas (Pekoe, Souchong and Congo) give clear, pale green to
yellowish green solutions. Teas not so black, or green teas,
give darker and moie brownish green solutions. In assaying
those varieties of tea, which probably contain a small quantity
of caffeine, take 12 grams and extract with 150 grams of
chloroform. C. C. Keller has shown that the best and most
expensive varieties of tea contain most caffeine. The average
percentage of caffeine, based upon 50 assays of tea, was found
to be 3.06. A green tea gave the smallest yield, namely,
1.78 per cent, of caffeine; and a Pekoe tea the highest yield,
namely, 4.24 per cent, of caffeine.
J. Gadamer states that Keller's method of estimating caffeine
in tea is appliqable also to coffee and cola preparations. Keller's
method is especially useful for roasted coffee. The caffeine,
though somewhat brown, is aways sufficiently pure.
274
DETECTION OF POISONS
2. Hilger-Juckenack Method. Macerate 20 grams of finely
ground coffee, or triturated tea, with 900 grams of water for
several hours in a large beaker at room temperatures. Then
boil thoroughly and replace the water lost by evaporation.
Raw coffee requires 3 hours and roasted coffee and tea 1.5
hours. Cool somewhat (60 to 80)
and add 75 grams of aluminum
acetate solution (see note, page
289) and gradually, while stirring,
1.9 grams of acid sodium carbon-
"N ate. Boil about 5 minutes and
bring the total weight when cold
to 1020 grams. Filter 750 grams
(=15 grams of original material)
and add to the clear nitrate 10
grams of precipitated and pow-
dered aluminum hydroxide and
some filter paper made into a
magma by agitation with water.
Stir frequently and evaporate
upon the water-bath. Thoroughly
dry the residue in an air-closet at
100, and extract for 8-10 hours
with pure tetrachloromethane
(CCU), using a Soxhlet apparatus
(Fig. 23). Tetrachloromethane,
which* is always colorless, is finally
distilled and the residue of per-
fectly white caffeine is dried at
100 and weighed. The results
thus obtained are usually accepted
without question. But if an absolutely accurate result is
required, nitrogen in the crude caffeine may be determined by
Kjeldahl's method. The quantity of anhydrous caffeine is
calculated on the basis of this analysis. One cc. of o.i n-oxalic
acid represents 0.00485 gram of anhydrous caffeine. Com-
mercial tetrachloromethane is usually impure and cannot be used
PIG. 23. Soxhlet Apparatus.
QUANTITATIVE ESTIMATION OF ALKALOIDS 275
directly in the extraction. It should be shaken 3-4 times with
sodium carbonate solution, then several times with water, dried
over fused calcium chloride and distilled fractionally. It boils
at 76-77.
3. Trfflich-Goeckel Modification of Hilger's Method
Exhaust 10 grams of finely ground coffee with water. This
will require 3 extractions with boiling water, using 200 cc.
portions and heating each for 30 minutes. Combine the filtered
extracts, cool and dilute to 495 cc. Add 5 cc. of basic lead ace-
tate solution, shake thoroughly, filter 400 cc. and pass hydrogen
sulphide through the filtrate. Dilute this filtrate to 500 cc.,
shake thoroughly and again filter 400 cc. This filtrate will
represent 6.4 grams of coffee. Concentrate this filtrate (400
cc.) upon the water-bath, and, after addition of i gram of
magnesium oxide and sand, evaporate to dryness. Triturate
the residue and extract for 30 hours with acetic ether, using a
Soxhlet apparatus. Evaporate the acetic ether extract in a
Kjeldahl flask, or distil the solvent, and determine nitrogen in
the residue by Kjeldahl's method. One gram of nitrogen rep-
resents 3.4643 grams of caff erne. Crude caffeine is easily
decomposed by the acid used hi the Kjeldahl process and by mer-
curic oxide. Roasted coffee may easily give too much caffeine
by this method, because the bases formed by roasting coffee,
pyridine for example, are also extracted by acetic ether.
4. Trillich-Goeckel Modification of Socolof's Method.
Put 10 grams of finely ground, dried coffee into a separating
funnel, provided with a plug of glass wool for a filter, and
moisten with ammonium hydroxide solution. Let the mixture
stand for 30 minutes, extract for 12 hours with 200 cc. of acetic
ether and shake frequently. Filter, and wash three times with
50 cc. portions of acetic ether. Distil the acetic ether upon the
water-bath and boil the residue with milk of magnesia. Filter,
and evaporate the filtrate to dryness upon the water-bath.
Dissolve the residual caffeine in acetic ether or chloroform.
Filter this solution into a weighed dish or Kjeldahl flask.
Evaporate the solvent and weigh the caffeine, or calculate it
from the percentage of nitrogen. The latter method is the more
276 DETECTION OF POISONS
accurate. According to C. Wolff 1 the residue from the acetic
ether or chloroform extract should not be accepted as pure caf-
feine. Determination of nitrogen in this residue by Kjeldahl's
method is the most reliable way of estimating caffeine in the
extract.
5. E. Kate's Method of Estimating Caffeine. This method
is based upon the fact that chloroform will extract caffeine
quantitatively from 'a solution which is ammoniacal, or faintly
acid with hydrochloric acid.
Shake 10 grams of powdered coffee, or tea, for 30 minutes
with 200 grams of chloroform and 5 grams of ammonium hy-
droxide solution. When the liquid has settled, filter 150
grams of the chloroform solution through a Sander's filter
which will give a perfectly bright filtrate free from water. Dis-
til the chloroform completely and dissolve the residue with
gentle heat in about 6 cc. of ether. Add 20 cc. of 0.5 per cent,
hydrochloric acid and, in an assay of coffee, also 0.2-0.5 gram
of solid paraffine. Evaporate the ether and filter the cold,
aqueous solution. Wash the flask and filter paper a few times
with small portions of 0.5 per cent, hydrochloric acid. Finally
extract the total aqueous hydrochloric acid solution four times
with 20 cc. portions of chloroform. Distil the filtered chloro-
form extract, dry the residue and weigh. This residue will
consist of nearly pure caffeine. J. Katz found the following
percentages of caffeine :
Caffeine Average
Raw Coffee Beans 0.9 -1.27 per cent. 1.14 per cent.
Dried Cola Nuts 1.51-1.94 per cent. 1.68 per cent.
Black Tea 2.51-3.56 per cent. 3.07 per cent.
Guarana 2.83-4.74 per cent. 4.08 per cent.
J. Katz recommends the following method for estimating
caffeine in mate or Paraguay tea:
Treat finely triturated tea with ammonium hydroxide solu-
tion and chloroform, as described above, and dissolve the chloro-
form residue in ether. Add water to the ether and evaporate.
Warm the aqueous solution 10 minutes upon the water-bath
1 Zeitschrift fur offentliche Chemie 12, 186.
QUANTITATIVE ESTIMATION OF ALKALOIDS 277
with 2 cc. of lead hydroxide suspended in water (i : 20). If it
is very difficult to get a clear nitrate from this liquid, add a
little calcined magnesium oxide. This treatment usually gives
a nitrate, which is perfectly clear when cold, and but slightly
colored. Chloroform extracts quite pure caffeine from this
solution. By this method mate yields 0.3-1.6 per cent, of
caffeine, the average being 0.71 per cent.
6. K. Dieterich's Method of Estimating Total Alkaloids
(Caffeine and Theobromine) in Cola Nuts. Moisten 10 grams
of finely grated cola nuts with a little water, mix with 10 grams
of granulated, unslaked lime and extract with chloroform in a
Soxhlet apparatus for 45 minutes. Evaporate the extract
almost to dryness and dissolve the residue with gentle heat
in 20 cc. of normal hydrochloric acid. Filter and dilute to 100
cc. Add ammonium hydroxide solution in large excess to this
nitrate, shake at frequent intervals during 15 minutes and
extract three times with 20 cc. portions of chloroform. Evapo-
rate this chloroform solution in a weighed flask and dry the
residue, which usually consists of perfectly pure caffeine, at 100
to constant weight.
This method may also be used in estimating caffeine in
Paraguay tea. Mix the finely ground material with unslaked
lime, and extract with chloroform, in a Soxhlet apparatus. Tea
gives pure, white caffeine free from chlorophyll.
Estimation of Alkaloids in Ipecac
Ipecac has been shown 1 to contain three alkaloids :
Cephaeline, C 2 8H4oN 2 O4, Emetine, Cso^NsO^
Psychotrine.
The composition of the last alkaloid is unknown. This drug
acts as an expectorant and emetic, because of cephaeline and
emetine. Psychotrine is said not to possess these properties.
Therefore, in assaying ipecac for medicinal purposes, only the
percentage of the first two alkaloids need be estimated. The
equivalent weights of these two alkaloids (cephaeline 234 and
1 Frerichs and de Fuentas Tapis, Archiv der Pharmacia, 1902, Heft 5 and 6.
278 DETECTION OF POISONS
emetine 248) are so nearly the same, that the mean of the two
(241) may be used as the factor.
Procedure. Put 6 grams of finely powdered root in a dry
Erlenmeyer flask and shake with 60 grams of ether. Then add
5 cc. of ammonium hydi oxide solution, or 5 cc. of sodium
carbonate solution (i 13), and shake frequently during an hour.
Add 10 cc. of water and, after shaking vigorously, filter 50
grams of the ether extract into a small flask. Evaporate half
the ether upon the water-bath, and extract the remainder in a
separating funnel with 10 cc. of o.i n-hydrochloric acid. Pass
the acid solution through a small filter into a 200 cc. flask.
Make two more extractions of the ether with 10 cc. portions of
.water, and pass these through the same filter. Bring the
volume of the acid solution to 100 cc., and then add enough
ether to form a layer about i cm. thick after thorough agi-
tation. Add 5 drops of iodeosine solution (i : 250), and titrate
excess of hydrochloric acid with o.i n-potassium hydroxide
solution. The number of cc. of o.i n-hydrochloric acid, com-
bined with the alkaloids, multiplied by 0.0241 gives the quan-
tity of emetine and cephaeline in 5 grams of ipecac.
To estimate -these alkaloids gravimetrically, shake vigorously
the ether solution of the alkaloids (50 grams = 5 grams of root)
in a separating funnel with 5 cc. of dilute hydrochloric acid
and 10 cc. of water. Transfer the acid solution to another
separating funnel. Make two more extractions of the ether
with 10 cc. portions of water and add these to the acid extract.
Add 5 cc. of ammonium hydroxide solution to the acid extract
and shake vigorously with 50 grams of ether. Remove the
aqueous layer and filter 40 grams of the ether solution into a
weighed flask. Evaporate the ether and weigh the flask after
drying for an hour at 100. This will give the quantity of eme-
tine and cephaeline in 4 grams of root.
Test for Cephaeline. This reaction is very characteristic of
this alkaloid. Froehde's reagent dissolves pure cephaeline, as
the free base, almost without color. A trace of hydrochloric
acid, or better sodium chloride, added to this solution produces
an intense blue color. Pure emetine gives no color with
QUANTITATIVE ESTIMATION OF ALKALOIDS 279
Froehde's reagent, nor when sodium chloride is added. This
test for cephaeline may be made with the ether residue.
The method of estimating alkaloids in ipecac, prescribed by the German
Pharmacopoeia, is the same as that for cinchona bark. Use 12 grams of finely
powdered root dried at 100, but in ascertaining excess of acid use iodeosine,
and not hsematoxylin, as the indicator. Finally, measure with a pipette 50
cc. of the proper solution having a volume of 100 cc., place in a 200 cc. flask
and add about 50 cc. of water and enough ether to make a layer i cm. thick.
Add 5 drops of iodeosine solution and enough o.oi n-potassium hydroxide
solution, shaking thoroughly after each addition, to give the lower aqueous
layer a pale red color. This should require not more than 20 cc. of alkaline
solution.
Estimation of Nicotine in Tobacco
i. R. Kissling's 1 Method. First remove the ribs and then
cut the tobacco leaves into small pieces. Dry 1-2 hours
(50-60), and then reduce to a uniform, coarse powder.
Triturate 20 grams of this powder with 10 cc. of dilute, alcoholic
sodium hydroxide solution (6 grams of sodium hydroxide
dissolved in 40 cc. of water and 60 cc. of 95 percent, alcohol).
Transfer this moist powder to a paper thimble and extract
2-3 hours with ether in a Soxhlet apparatus. Carefully distil
the ether solution so that a portion of the solvent remains. Add
50 cc. of very dilute sodium hydroxide solution (4 grams of
sodium hydroxide in 1000 cc. of water) to the residue- and distil
with steam. Begin introducing steam after the nicotine solu-
tion has been boiling several minutes. Collect about 400 cc. of
distillate, that is to say, continue distilling until the distillate is
no longer alkaline. Mix well, add a few drops of rosolic acid
solution to the distillate, and titrate nicotine with o.i n-sul-
phuric or oxalic acid until the red color has just disappeared.
Calculation. Although nicotine, Ci Hi 4 N 2 (162), as a di-acid
base can combine with two equivalents of acid, it behaves upon
titration, with rosolic acid or iodeosine as indicator, as if^it
were a monacid base with the equivalent weight 162. 1000 cc.
of o.i n-acid consequently correspond to 16.2 grams of nicotine.
2. C. C. Keller's 2 Method. Pour 60 grams of ether and 60
grams of petroleum ether over 6 grams of dry tobacco in a 200
1 Zeitschrift fur analytische Chemie 34, 1731 and 21, 76.
2 Berichte der Deutschen pharmazeutischen Gesellschaft 8, 145 (1898).
280 DETECTION OF POISONS
cc. Erlenmeyer flask. Add 10 cc. of 20 per cent, aqueous
potassium hydroxide solution and let the mixture stand half an
hour, shaking vigorously at frequent intervals. After the liquid
has stood at rest 3-4 hours, pour 100 grams of ether solution
through a small, plaited filter and receive the filtrate in a 200 cc.
Erlenmeyer flask. Nicotine is in solution together with a little
ammonia, which must be removed before titration. By means
of a hand bellows and a glass tube reaching to the bottom of the
flask force a current of air through the solution, so that there
is considerable agitation. It requires about a minute and
a half to expel all ammonia. At the same time 8-10 grams
of ether evaporate. Add 10 cc. of alcohol, a drop of i per
cent, iodeosine solution and 10 cc. of water to the ammonia-
free solution. Stopper the flask and shake vigorously.
Nicotine and iodeosine dissolve in the water which has a
red color. Add a slight excess of o.i n- hydrochloric acid,
enough to discharge the color, and titrate excess of acid with
o.i n-potassium hydroxide solution. The quantity of nicotine
in tobacco shows a wide variation and ranges from 0.6 to 4.8
per cent.
3. J. Toth's 1 Method. According to Toth two sources of
error in C. C. Keller's method lead to low results. An aqueous
potassium hydroxide solution retains variable quantities of
nicotine and a current of air passed through an ether solution
of nicotine volatilizes some of this alkaloid. Therefore Toth
recommends the following procedure:
Mix 6 grams of air-dried tobacco with 10 cc. of 20 per cent,
sodium hydroxide solution in a porcelain dish. Add gypsum
until the mixture is like powder. Extract thoroughly with 100
cc. ether-petroleum ether mixture (i : i) and after i hour pipette
off as quickly as possible 25 cc. of the solvent. Add 40-50 cc.
of water, i drop of iodeosine solution and an excess of o.i n-sul-
phuric acid. Determine excess of acid by titration with o.i
n-sodium hydroxide solution. The ether-petroleum ether
mixture takes up at most 0.0005 gram of ammonia.
1 Chemisches Zentralblatt, 1901, i, 973.
QUANTITATIVE ESTIMATION OF ALKALOIDS 281
Estimation of Hydrastine in Fluid Extract of Hydrastis
(German Pharmacopoeia)
Evaporate 15 grams of fluid extract of hydrastis to about 5 grams in a weighed
dish upon the water-bath, and wash the residue into an Erlenmeyer flask with
about 10 cc. of water. Add 10 grams of petroleum ether, 50 grams of ether and 5
grams of ammonium hydroxide solution. Let the mixture stand an hour, shaking
vigorously at frequent intervals. Then pass 50 grams of the clear ether solution
through a dry filter into a separating funnel. Add 10 cc. of a mixture, composed
of i part of hydrochloric acid and 4 parts of water, and shake the solution
vigorously several minutes. When the liquids have separated clear, run the
acid solution kito an Erlenmeyer flask. Make two more extractions of the ether
with 5 cc. portions of water containing a few drops of hydrochloric acid, and add
these to the first extract. Add to the total extract excess of ammonium hydrox-
ide solution and 50 grams of ether. Let the mixture stand an hour, shaking
vigorously at frequent intervals. Pass 40 grams of the clear ether solution
through a dry filter and collect the filtrate in a weighed, dry flask. Distil the
ether, dry the residue at 100 and weigh when cold. The residue should weigh at
least 0.2 gram.
Notes. Additional information about hydrastine is given
on page 116. Ammonia, added to an aqueous solution of the
residue from hydrastis extract (15 grams), sets the alkaloids,
hydrastine and berberine, free from their salts. The ether-
petroleum benzine mixture dissolves hydrastine but not ber-
berine, the latter being nearly insoluble in this mixed solvent.
But phytosterin, which is always present in hydrastis extract, is
dissolved. Only 50 grams (= hydrastine in 12.5 grams of
extract) of the original 60 grams of ether-petroleum benzine
mixture are used. Hydrastine is extracted from the solvent by
agitation with dilute hydrochloric acid and dissolved in the
acid solution as hydrochloride. The alkaloid is then precipi-
tated from the acid solution by ammonia and dissolved in 50
grams of ether:
C 21 H 21 N0 6 .HC1 + (H 4 N)OH = C 2 iH 21 NO 6 + H 2 O + (H<N)C1.
Hydrastine Hydrastine
hydrochloride
The hydrastine in 40 grams of the ether solution (= 10 grams
of original extract) is finally weighed. Good extract of hydras-
tis should contain 2-2.5 P er cent - f hydrastine.
When the ether-petroleum benzine solution of hydrastine and
phytosterin is extracted with dilute hydrochloric acid, the
alkaloid passes into the acid solution free of phytosterin.
282 DETECTION OF POISONS
Estimation of Berberine. This alkaloid has only a slight physiological action.
To determine approximately the quantity present in hydrastis extract, add 20
grams of dilute sulphuric acid (i : 5) to 10 grams of the extract and let the mixture
stand for 24 hours at as low a temperature as possible. Crystallization of ber-
berine as the difficultly soluble acid sulphate, CzoHnNO^H^SO^ is almost com-
plete. Filter in a Gooch crucible with suction, washing, first with a little water
containing sulphuric acid and then with pure water. Dry at 100 to constant
weight. (E. Schmidt.)
W. Meine 1 has found that the crystalline deposit, frequently seen in hydrastis
extract, consists mostly of berberine mixed with a little phytosterin. This
deposit is said to contain only traces of hydrastine.
Picrolonate Method of Estimating Hydrastine in Hydrastis Root and
Extract
(H. Matthes and 0. Rammstedt) 2
The German Pharmacopoeia requires the estimation of
hydrastine but not of the physiologically inert substances,
berberine and phytosterin, also present in hydrastis prepara-
tions. Ether-petroleum benzine mixture, used as a solvent,
dissolves phytosterin and hydrastine but not berberine. Di-
lute hydrochloric acid extracts hydrastine but leaves phytos-
terin in the ether mixture.
Estimation of hydrastine by means of picrolonic acid appears
simpler than by the method of the Pharmacopoeia, because
picrolonic acid does not precipitate phytosterin and therefore
hydrastine is not mixed with this impurity. Matthes and
Rammstedt obtained nearly pure hydrastine picrolonate from
hydrastis extract, melting at 220-225. The picrolonate pre-
pared from pure hydrastine, C2iH2iNO6.CioH 8 N4O 6 , melts at
225.
i. In Fluid Extract of Hydrastis. Evaporate 15 grams of
fluid extract to about 5 grams in an Erlenmeyer flask upon the
water-bath. Add 10 cc. of water; 10 grams of petroleum
benzine, 50 grams of ether and 5 grams of ammonium hydroxide
(10 per cent. NHs). Shake vigorously for 10 minutes. After
the mixture has stood for 20 minutes, pour 40 grams of the ether-
1 Zeitschrift des allgemeinen osterreichischen Apotheker-Vereins 55, 494.
8 Further information about picrolonic acid and its use in precipitating alka-
loids is given on page 253.
QUANTITATIVE ESTIMATION OF ALKALOIDS 283
benzine extract through a double, creased filter and evaporate
about one-half in a beaker. Then add 10 cc. of o.i n-picrolonic
acid solution. After 24 hours collect the hydrastine picrolonate
in a weighed Gooch crucible, wash with 2 cc. of an alcohol-ether
mixture (i 13), dry for 30 minutes at 105 and weigh.
2. In Hydrastis Root. Shake 6 grams of powdered root
vigorously for 30 minutes with 50 grams of ether, 10 grams of
petroleum ether and 6 grams of ammonium hydroxide (10 per
cent. NH 3 ). Then add 6 grams of water and shake until the
upper layer of liquid is clear. Quickly filter 50 grams of the
ether-petroleum benzine extract and evaporate about one-half
in a beaker. Then add 5 cc. of o.i n-picrolonic acid. After 24
hours filter the picrolonate precipitate and wash with i cc. of
alcohol-ether mixture (i 13). Otherwise the procedure is the
same as described in i.
In the calculation use the formula of hydrastine picrolonate
(Mol. Wt. 647) given above.
Estimation of Morphine in Opium and Pharmaceutical Preparations
(German Pharmacopoeia)
In Opium. Triturate 6 grams of rather finely powdered opium with 6 grams of
water. Wash the mixture into a weighed, dry flask with water and add enough
more of this solvent to bring the weight to 54 grams. Shake frequently and let
the mixture stand an hour. Pour upon a piece of dry linen and express the
liquid. Pass 42 grams of this extract through a dry, plaited filter (10 cm. in di-
ameter) into a dry flask. Add 2 grams of sodium salicylate solution (i : 2) to this
nitrate and shake vigorously. Filter 36 grams of the clear solution through a dry,
plaited filter (10 cm. in diameter) into a small flask. Mix this filtrate by gentle
agitation with 10 grams of ether, and add also 5 grams of a mixture consisting of
1 7 grams of ammonium hydroxide solution and 83 grams of water. * Stopper the
flask, shake vigorously for 10 minutes and let the mixture stand at rest 24 hours.
Then decant the ether layer as completely as possible upon a smooth filter
(8 cm. in diameter). Add 10 grams more of ether to the residual, aqueous liquid
in the flask, shake gently for a few minutes and again pour the ether layer upon
the filter. Then after all the ether solution has passed through, pour the aque-
ous solution upon the filter, and disregard crystals adhering to the side of the
flask. Wash filter and flask three times with 5 cc. portions of water saturated
with ether. When the filter has drained thoroughly, dry the morphine crystals
and dissolve in 25 cc. of o.i n-hydrochloric acid. Pour this solution into a
100 cc. flask, carefully wash filter and flask with water and finally dilute the
solution to 100 cc. Measure 50 cc. of this solution into a 200 cc. flask, add
284 DETECTION OF POISONS
50 cc. of water and enough ether to form a layer i cm. thick. Add 5 drops of
iodeosine solution and enough o.i n-potassium hydroxide solution, shaking vigo-
rously after each addition, to produce a pale red color in the lower aqueous layer. 1
Notes and Calculation. Most of the opium alkaloids are
combined with meconic (see page 213) and sulphuric acids.
Ammonium hydroxide, added to an aqueous opium extract,
sets the alkaloids free from their salts:
(Ci7Hi9NO 8 )2 OH
CsH0 2 (OH)(COOH) 2 + 2(H 4 N)OH = 2 C 17 H lfl NO 3 + 2H 2 O + C 5 HO 2
(COONH 4 ) 2
Morphine meconate Morphine Ammonium
meconate
The ether used dissolves all opium alkaloids except morphine
which having once become crystalline is insoluble in this sol-
vent. Saturated sodium salicylate solution precipitates resinous
and greasy substances from the filtered aqueous opium extract
and also narcotine which next to morphine is present in opium
in largest quantity.
The morphine from 6 grams of opium is in 54 grams of fil-
tered aqueous extract. After the second filtration only 3 6 grams
of this extract are used (= morphine from 4 grams of opium).
Morphine, precipitated by ammonium hydroxide from these 36
grams of extract, is dissolved in 25 cc. of o.i n-hydrochloric acid
as hydrochloride, CnHigNOs.HCl. This solution is then diluted
to 100 cc. and excess of acid in 50 cc. of this hydrochloric acid
solution (= morphine from 2 grams of opium) is determined.
Morphine being a monacid base has the same molecular and
equivalent weights = Ci 7 Hi 9 NO 3 = 285. Therefore 1000 cc.
of o.i n-hydrochloric acid = 28.5 grams of morphine.
Example. Titration with o.i n-potassium hydroxide solution has shown that
there are 4.1 cc. of o.i n-hydrochloric acid in 50 cc. of the hydrochloric acid
solution of morphine. There remain therefore 12.54.1 =8.4 cc. of the/xi
n-acid originally present now combined with the morphine from 2 grams^of
opium. According to the proportion
Cc. o.i n-HCl : Grams morphine
1000 : 28.5 = 8.4 : x (x = 0.2394)
1 The German Pharmacopoeia demands that not more than 5.4 cc. nor less than
4.1 cc. of o.i n-alkaline hydroxide solution, corresponding to a morphine-content
of 10-12 per cent., shall be used to produce this color.
QUANTITATIVE ESTIMATION OF ALKALOIDS 285
8.4 cc. of o.i n-acid correspond to 0.2394 gram of morphine. Consequently the
opium contains 50 X 0.2394 = 11.97 per cent, of morphine. This is the maxi-
mum quantity of morphine allowed in opium by the German Pharmacopoeia.
2. In Extract of Opium. "Dissolve 3 grams of opium extract in 40 grams of
water, add 2 grams of sodium salicylate solution (1:2), shake vigorously, pass 30
grams of clear solution through a dry filter (10 cm. in diameter) and collect
in a dry flask. Mix this nitrate with 10 grams of ether by rotating the flask and
add also 5 grams of a mixture of 17 grams of ammonium hydroxide and 83 grams
of water." Continue the assay as directed above in i (Opium) from the point
marked with an asterisk.
Calculation. Only 2 of the 3 grams of opium extract weighed are used in the
determination, since only 30 grams of the original 45 grams of solution (3 grams
of extract + 2 grams of sodium salicylate solution + 40 grams of water) are
filtered. The morphine obtained from these 2 grams of extract is dissolved in 25
cc. of o.i n-hydrochloric acid and the volume is then brought to 100 cc. The
titration uses 50 cc. of this solution which contains the morphine from x gram of
opium extract.
Example. If 5.5 cc. of o.i n-potassium hydroxide solution were required to
neutralize the excess of o.i n-hydrochloric acid in the 50 cc. of solution, then
12.5 5.5 = 7 cc. of o.i n-hydrochloric acid are combined with morphine. Ac-
cording to the proportion
Cc. o.i n-HCl : Grams morphine
1000 : 28.5 = 7 : x (x = 0.1995)
7 cc. of o.i n-acid correspond to 0.1995 gram of morphine. Therefore this
quantity of alkaloid is in i gram of extract. Consequently the opium extract
contains 19.95 per cent, of morphine.
The German Pharmacopoeia requires that not more than 6.5 cc. nor less than
5.5 cc. of o.i n-potassium hydroxide solution shall be used to produce a pale
red color in the aqueous layer, corresponding to a morphine content of 17.11 to
19.95 per cent.
3. In Wine of Opium and Tincture of Opium. " Evaporate about 50 grams
of either preparation in a weighed dish to 15 grams, add water until the weight
is 38 grams and also 2 grams of sodium salicylate solution (i : 2). Shake vig-
orously and pass 32 grams of clear solution through a dry creased filter (10 cm.
in diameter) into a dry flask. Mix this filtrate with 10 grams of ether by rotating
the flask and add also 5 grams of a mixture of 17 grams of ammonium hydroxide
solution and 83 grams of water." Continue the assay as directed above in i
(Opium) from the point marked with an asterisk.
Calculation. Only 32 grams of the 40 grams of clear liquid (38 grams of evapo-
rated opium tincture + 2 grams of sodium salicylate solution) are used for the
morphine determination. These correspond to 40 grams of the original opium
preparation. The morphine from this quantity of solution is dissolved in 25
cc. of o.i n-hydrochloric acid and the volume brought to 100 cc. Excess of acid
in 50 cc. of this solution is determined by titration. These 50 cc. contain the
morphine from 20 grams of the opium preparation.
Example. If 4.2 cc. of o.i n-potassium hydroxide solution are required for
50 cc. of morphine hydrochloride solution, then 12.5 - 4-2 = 8.3 cc. of o.i n-
286 DETECTION OF POISONS
hydrochloric acid have combined with morphine. According to the proportion
Cc. o.i n-HCl : Grams morphine
looo : 28.5 = 8. 3 : x (x = 0.23655)
20 grams of the opium preparation contain 0.23655 gram of morphine, corre-
sponding to a morphine content of 1.18 per cent.
The German Pharmacopoeia requires that not more than 5.55 cc. nor less than
4.2 cc. of o.i n-potassium hydroxide solution shall be used to produce a pale
red color in the aqueous liquid, corresponding to a morphine content in Wine
of Opium and Tincture of Opium of i.o to 1.18 per cent.
Estimation of Pilocarpine in Jaborandum Leaves 1
1. G. Fromme's 2 Method. Extract 15 grams of rather finely
powdered Jaborandum leaves with 150 grams of chloroform and
15 grams of ammonium hydroxide solution (10 per cent. NHs),
shaking frequently for 30 minutes. Filter this mixture through
a large, smooth paper, covering the funnel with a glass plate.
As soon as the chloroform drops slowly, add a little water and
filtration will become more rapid. After collecting a full 100
grams of nitrate, add about i gram of water, shake vigorously
and set aside. The water takes up fine particles of powder
that may have passed through the paper, leaving the chloro-
form solution quite clean. After i hour weigh 100 grams of
chloroform solution (= alkaloids in 10 grams of Jaborandum
leaves).
Fromme directs extracting these 100 grams of chloroform
solution successively with 30, 20 and 10 cc. of i per cent, hydro-
chloric acid which dissolves pilocarpine (and isopilocarpine) as
hydrochlorides. Extract this acid solution first with 20 cc. of
ether, to remove fat and resin. Then add an excess of ammonia
and extract the free alkaloid 5 successively with 30, 20 and 10 cc.
of chloroform. Pour the combined chloroform extracts through
a dry filter, evaporate in a weighed flask, dry the residue at 100
and weigh.
2. Matthes and Rammstedt's 3 Method. Evaporate 100
grams of chloroform solution obtained above in a beaker to
1 Further information about pilocarpine is given on page 217.
2 Caesar and Loretz, Geschaftsbericht 1901, 27.
3 See page 253.
QUANTITATIVE ESTIMATION OF ALKALOIDS 287
about 20 cc. Add first 3 cc. of o.i n-picrolonic acid and then
60 cc. of ether. After 24 hours collect the precipitate of pilo-
carpine picrolonate in a weighed Gooch crucible, wash with i cc.
of alcohol-ether mixture (i : 3), dry at 110 and weigh. Pilo-
carpine picrolonate thus obtained (= pilocarpine from 10
grams of jaborandum leaves), CiiHi 6 N 2 O 2 .CioH 8 N4O5 (Mol.
Wt. 472) melts at 200-205.
Piperine in Pepper
Black pepper is the dried, unripe fruit of the pepper plant, Piper nigrum L.,
whereas the ripe fruit deprived of its outer covering is the white pepper of com-
merce. The actual constituents of pepper are piperine, an ethereal oil (oil of
pepper) and a resin called chavicine. In rather large doses pepper is toxic. 1
Preparation of Piperine. Extract finely divided white pepper with oo per cent,
alcohol and distil the latter from the extracts. Treat the residue with cold
potassium hydroxide solution which dissolves the resin but not the piperine.
Wash the residual piperine with water and crystallize from hot alcohol, using
animal charcoal to remove color. White pepper contains 7-8 per cent, of
piperine.
Piperine, CnHjgNOs, crystallizes in colorless, shining, rectangular, monoclinic
prisms melting at 1 28-1 29. When pure it is almost tasteless but impure piperine
has a sharp, burning taste. It is nearly insoluble in water, freely soluble in alco-
hol and also soluble in ether, benzene and chloroform. Piperine is a very weak
base, dissolving in dilute mineral acids with almost as much difficulty as in pure
water. A solution of piperine in concentrated sulphuric acid has a ruby color,
soon changing to dark brown and gradually to greenish brown and fading upon
addition of water. Concentrated nitric acid converts piperine into an orange-
red resin soluble with blood-red color in dilute potassium hydroxide solution.
The constitution of piperine is known.
Prolonged heating with alcoholic potassium hydroxide solution decomposes
piperine into the potassium salt of piperic acid and piperidine :
CH = CH.CO.NC6H 10 CH = CH.COOK
+ KOH = C5H 10 NH + |
CH = CH.C 6 H 3 (0 2 CH 2 ) Piperidine CH = CH.C 6 H 3 (OCH 1 )
Piperine Potassium piperate
Riigheimer 1 synthesized piperine by putting together these two products.
Piperic acid was first converted into its chloride by means of phosphorus penta-
J R. Kobert ("Intoxikationen") mentions a. case where a teaspoonful of
pepper was given to each of three young pigs. There was severe inflammation
of the gastro-intestinal tract in all three animals and two died. The toxic actio
of pepper is attributed to piperine, since the ethereal oil according to Kobert does
not take part in the toxic action due to absorption.
2 Berichte der Deutschen chemischen Gesellschaft 15, 139 (1882).
288 DETECTION OF POISONS^
chloride. Piperyl chloride was then condensed in benzene solution with piperi-
dine:
CH = CH.COOH CH = CH.COC1
| +PC1 5 = I +POC1 3 +HC1
CH = CH.C 6 H 3 (0 2 CH 2 ) CH = CH.C 6 H 3 (0 2 CH 2 )
CH = CH.COC1 CH = CH.CO.NC 6 H 10
| +HNC 5 H 10 =| +HC1.
CH = CH.C 6 H 3 (0 2 CH 2 ) CH = CH.CeH 3 (0 2 CH 2 )
Piperyl chloride Piperidine Piperine
On the basis of the known structure of piperic acid and piperidine, piperine
must have the following constitution:
H H 2
C C
/\ /\
/O C CH H 2 C CH 2
H 2 C< | || ||
X C CH H 2 C CH 2
C N
CH=CH.CH=CH.CO
Estimation of Piperine in Pepper
1. J. Koenig's Method. Exhaust 10-20 grams of pepper,
ground as finely as possible, in a Soxhlet apparatus with strong
ethyl or methyl alcohol, or petroleum ether. Distil the alcohol
or petroleum ether. The residue consists of piperine and resin.
Shake this residue with cold potassium or sodium carbonate
solution to dissolve the resin. Filter from undissolved piperine
and wash the latter with cold water. Dissolve in alcohol or
petroleum ether, evaporate the filtered solution in a weighed
flask or dish and dry the residue at 100 to constant weight.
To determine the resin in pepper at the same time, filter the
potassium or sodium carbonate solution from crude piperine and
add hydrochloric acid to the filtrate. Filter the precipitated
resin, redissolve in alcohol, evaporate the solvent and dry the
residue to constant weight.
2. Cazeneuve and Caillof s Method. Add enough water to
make a thin mixture of powdered pepper with twice its weight
of slaked lime and stir well. Boil in a porcelain dish, dry thor-
oughly upon the water-bath and then extract with ether in a
Soxhlet apparatus. Distil the ether in a weighed flask and
QUANTITATIVE ESTIMATION OF ALKALOIDS 289
dry the residue of piperine at 100 to constant weight. To
obtain pure crystalline piperine, dissolve the residue from the
ether distillation in the least possible volume of boiling alcohol,
surround the solution with ice, collect the piperine upon a
weighed filter and dry at 100 to constant weight. This
purification of piperine is attended with more or less loss and
consequently the result is only approximately correct.
Estimation of Santonin in Wormseed 1
i. K. Thaeter's 2 Method. Extract 10 grams of crushed
wormseed in a Soxhlet apparatus with ether for 12 hours.
Distil the ether and boil the residue for an hour with 5 grams
of lime and about 300 cc. of water. Replace water lost by
evaporation. Filter while hot and wash the residue with
water. Faintly acidify the filtrate with sulphuric acid and
warm gently until santonin crystals begin to form. Then add
100 grams of aluminium acetate solution, 3 heat the mixture
to boiling and finally evaporate to dryness upon the water-
bath. Mix the finely powdered residue with 3 grams of mag-
nesium oxide, moisten this mixture with a little water and again
bring quickly to dryness. Powder the residue as finely as pos-
sible, dry at 105 and extract in a Soxhlet apparatus with an-
hydrous, acid-free ether for 5 hours. Santonin is deposited
upon distilling the ether as a faintly yellowish residue which
is then dried at 100 to constant weight.
Remarks. When wormseed is heated with lime, santonin passes into solution
as calcium santonate, and at the same time resinous substances are. saponified.
1 Wormseed (Flores cinas) consists of the unexpanded flower-heads of Artemisia
cina which are 3-4 mm. in length.
z Archiv der Pharmacie 237, 626-632 (1899) and 238, 383-387 (1900).
3 Dissolve 300 parts of aluminium sulphate in 800 parts of water; add acetic
acid (sp. gr. 1.041) 360 parts; triturate calcium carbonate 130 parts with 200
parts of water, and add this mixture slowly and with continued stirring to the
first solution; set the whole aside for 24 hours without applying heat, and stir
occasionally; then strain, press the precipitate without washing it and filter the
liquid. It is a clear, colorless liquid, having the sp. gr. 1.044 to 1.046, a faint
odor of acetic acid, an acid reaction, and a sweetish, astringent taste. National
Dispensatory.
290 DETECTION OF POISONS
Dilute sulphuric acid liberates first santonic acid which passes at once into its
inner anhydride, santonin. Basic aluminium acetate, produced by boiling,
precipitates resinous and colored substances. Finally, magnesium oxide serves
to neutralize free acetic acid. Under the conditions, practically no magnesium
santonate is formed. Thaeter obtained 88 to 92 per cent, of the santonin present.
Wormseed contains about 2.5 per cent, of santonin.
2. J. Katz's 1 Method. Extract 10 grams of coarsely pow-
dered wormseed in a Soxhlet apparatus with ether for 2 hours.
Distil the ether. There usually remains a dark green resin
weighing 1.5-2 grams. Boil this residue 1 5-30 minutes, under a
reflux condenser, with 5 grams of crystallized barium hydroxide
dissolved in 100 cc. of water. Cool and, without filtering,
render the solution acid to litmus with carbon dioxide. Filter
immediately with a pump to remove barium carbonate, and
wash the precipitate twice with 20 cc. portions of water. Evap-
orate the pale yellow solution to about 20 cc. in a dish upon the
water-bath. Add 10 cc. of 12.5 per cent, hydrochloric acid,
continue heating upon the water-bath exactly 2 minutes longer
and pour the solution into a separating funnel. Dissolve
santonin crystals left in the dish in about 20 cc. of chloroform.
Pour this solution into the separating funnel and shake thor-
.oughly. When the solutions have separated clear, withdraw
the chloroform solution and pass it through a dry filter. Wash
dish, separating funnel and filter 2-3 times with 10 cc. portions
of chloroform. Distil the chloroform and boil the residue 10
minutes, under a reflux condenser, with 50 cc. of 15 per cent,
alcohol. Filter while hot into a weighed flask, and wash flask
and filter twice with 15 cc. portions of 15 per cent, boiling
alcohol. Cover the flask and set aside in the cold 24 hours.
Weigh flask and contents and pass the latter through a weighed
filter, disregarding the milky appearance of the filtrate caused
by minute, resinous drops. Wash flask and filter once with 10
cc. of 15 per cent, alcohol, dry the filter in the flask and weigh
both. Finally, apply a correction on account of the solubility of
santonin in the alcohol used. Every 10 grams of filtrate contain
6 mg. of santonin. Santonin by this method is crystalline,
1 Archiv der Pharmacie 237, 251 (1899).
QUANTITATIVE ESTIMATION OF ALKALOIDS 291
and usually faintly yellow. J. Katz found the quantity of
santonin in wormseed to vary between 1.21 and 3.16 (average
2.42) per cent. This method is based upon the fact that the
santonin in 10 grams of wormseed is easily soluble in 50 cc. of
hot 15 per cent, alcohol, whereas only a very little resin is dis-
solved by this dilute alcohol. As this dilute, alcoholic solution
cools, santonin crystallizes out almost quantitatively.
Troches of Santonin. To estimate the- quantity of santonin in troches, made
from this substance and sugar, directly extract the finely ground mixture
with hot chloroform. The santonin can usually be weighed without further
purification. .
Chocolate Troches of Santonin. In a somewhat simpler form, the method
described above may be used to estimate santonin in chocolate troches. Weigh
3 or 4 troches and boil 15 minutes under a reflux condenser with 5 grams of barium
hydroxide and 100 cc. of water. Saturate the liquid when cold with carbon
dioxide. Filter, wash the residue with water and evaporate the brownish filtrate
to loo cc. Warm the liquid and add 10 cc. of dilute hydrochloric acid. Three
extractions with chloroform yield nearly pure santonin. To get santonin crys-
tals almost white and ready for weighing, evaporate the chloroform solution and
expel the last traces of chloroform by adding a few cc. of ether. If santonin is
impure from traces of fatty acids, boil once with 10 cc. of petroleum ether and
filter when cold. Santonin is nearly insoluble in cold petroleum ether.
Santonin can be detected and estimated in lexicological analysis in a similar
manner. Acidify the material with hydrochloric acid, extract with chloroform
and treat the chloroform residue with barium hydroxide solution as described
above.
Estimation of Solanine in Potatoes 1
i. O. Schmiedeberg and G. Meyer's Method. Mix 500
grams of finely grated potatoes with water and press out the
liquid. Decant the liquid from the deposit of starch. Again
mix the starch with water and decant the latter when the
starch has settled. Neutralize the entire liquid with ammonia
and evaporate to the consistency of an extract. In the mean-
time mix the press-cake with several times its volume of boiling
alcohol. Press out the alcohol completely after several hours.
Make two such extractions. Filter the combined alcoholic
extracts and wash the residue (starch) upon the filter with
alcohol. The aqueous liquid from the potatoes contains very
1 See page 225.
292 DETECTION OF POISONS
little solanine. To isolate this small quantity, use the alcoholic
filtrate to extract the residue from the aqueous extract and
again filter. Wash the insoluble part with hot alcohol. The
alcoholic filtrate after half an hour usually deposits some
crystals of asparagine. 1 Separate the supernatant liquid from
these crystals and evaporate upon the water-bath to the con-
sistency of an extract. Dissolve the residue in water con-
taining sulphuric acid, filter and wash. Warm the clear liquid
very gently, saturate with ammonia and set aside for a day.
Solanine appears in small crystals. Collect the deposit upon
a weighed filter, wash first with water and then with ether,
dry at 100 and weigh.
2. F. von Morgenstern's 2 Method. Express as much liquid
as possible from 200 grams of finely grated potatoes by means
of a press. Make two separate extractions of the press-cake
with water and express the liquid thoroughly each time. Pre-
cipitate protein substances from the combined liquid by add-
ing 0.5 cc. of acetic acid and warming for i hour upon the
water-bath. Filter, evaporate the filtrate to a syrup, stir and
add gradually hot 96 per cent, alcohol until cloudiness ceases. 3
Decant the solution after 12 hours and extract the residue
containing sugars and dextrins twice with hot alcohol. Evapo-
rate the combined alcoholic extracts upon the water-bath, warm
the residue with some water containing acetic acid and filter.
Heat the filtrate to boiling and add ammonia drop by drop to
precipitate solanine. After standing for 5 minutes upon the
water-bath, the base separates in flocks that are easily filtered.
Wash the precipitate with water containing ammonia, dissolve
in boiling alcohol and treat this solution as follows. Evaporate
1 Asparagine is the amide of aspartic acid, or mono-amino-succinic acid,
H 2 N.CH COOH
+ HaO. It appears in shining, rhombic crystals that
CHz CO .NH 2
dissolve rather easily in hot water but less easily in alcohol or ether. Laevo-
asparagine is widespread in the plant kingdom in seeds.
2 Landwirtschaftliche Versuchsstation 65, 301 (1907).
3 To extract those parts of the potato plant, which can be dried at 100 and
reduced to a fine powder, heat to boiling several times with water containing
acetic acid and filter each time.
QUANTITATIVE ESTIMATION OF ALKALOIDS 293
upon the water-bath and dissolve the residue in water containing
acetic acid. Filter, heat the nitrate to boiling and precipitate
solanine with ammonia. Collect the pure white flocks of sola-
nine from this second precipitation upon a filter that has been
dried at 90 and weighed. Wash with 2 per cent, ammonia and
dry at 90 to constant weight.
Notes. v. Morgenstern obtained on the average by this method 0.0125 per
cent, of solanine in table potatoes and 0.0958 per cent, in those used as forage.
The yield of solanine from yellow tubers upon the average was less than from red.
Tubers grown upon sandy soil were richer in solanine than were those from humus
soil. Moisture and abundance of humus appear to diminish the quantity of
solanine. A nitrogenous fertilizer increased the quantity of solanine, a potash
fertilizer lowered it and a phosphate fertilizer appeared to have little effect.
There was less solanine in large than in small potatoes of the same variety.
Solanine first appears to increase during the process of germination. Passing
into sprouts, without wholly disappearing from the tubers, solanine increases
with the growth of the plant. As growth advances the distribution of solanine
in the different parts of the plant is indication of a tendency on the part of the
plant to withdraw solanine from the older sprouts and spread it throughout the
young organs. Consequently solanine may serve first of all as the natural
protector of the plant, especially of the growing parts.
Estimation of Alkaloids in Nux Vomica
(C. C. Keller 1 )
Remove fat from nux vomica by treating 15 grams of the
well-dried and finely powdered drug in a 250 cc. Erlenmeyer
flask two or three times with 30 cc. portions of ether. Shake
thoroughly for 5 minutes. Pour these ether washings into a
flask, and, since they contain a little alkaloid, extract dissolved
alkaloid with 5 cc. of o.i n-hydrochloric acid and 10 cc. of water.
Repeat the extraction of the ether layer, separated from the
aqueous solution, using water instead of acid. Add 100 cc. of
ether, 50 grams of chloroform and 10 grams of 10 per cent, am-
monium hydroxide solution to the powdered nux vomica free
from fat. Shake thoroughly for 30 minutes and add to this
mixture the hydrochloric acid solution used in extracting alka-
1 Festschrift presented at the fiftieth anniversary of the founding of the Swiss
Pharmaceutical Association. Abstract in Zeitschrift fur analytische, Chemie,
23, 491 (1894).
294 DETECTION OF POISONS
loid from the first ether washings. Again shake thoroughly
and, when the liquids have separated clear, pour 100 grams of
ether-chloroform solution through a small filter into a weighed
Erlenmeyer flask. Distil the chloroform and ' ether as com-
pletely as possible. The alkaloids usually appear as colorless
varnishes which persistently retain chloroform. To remove the
latter, pour a few cc. of absolute alcohol upon the residue and
expel completely upon the water-bath. Repeat this treatment
2-3 times. This will give crystalline alkaloids which can be
dried at 100 to constant weight.
Method of the German Pharmacopeia
i. In Nux Vomica. Place 15 grams of nux vomica, ground mediumly fine
and dried at 100, in an Erlenmeyer flask and add 100 grams of ether and 50
grams of chloroform. Shake vigorously and add 10 cc. of a mixture of 2 parts
of sodium hydroxide solution and i part of water. Shake at frequent intervals
and let the mixture stand for 3 hours. Then add 15 cc. more water, or enough
to cause the powder after vigorous shaking to gather into balls and leave the
supernatant ether-chloroform solution perfectly clear. After i hour filter 100
grams of the clear ether-chloroform solution through a dry filter kept well
covered. Collect the filtrate in a small flask and distil about half the solvent.
Transfer the residual ether-chloroform solution to a separating funnel, rinse the
flask 3 times with 5 cc. portions of a mixture of 3 parts of ether and i part of
chloroform. Extract the combined solvent with 10 cc. of o.i n-hydrochloric
acid. Add enough ether to cause the ether-chloroform solution to rise to the top
of the acid liquid and pass the latter through a small filter moistened with water
into a zoo cc. flask. Then extract the ether-chloroform solution with 3 addi-
tional 10 cc. portions of water. Pass these extracts through the same filter,
wash the latter with water and dilute the total liquid to ico cc. Finally measure
50 cc. of this solution into a flask holding about 200 cc., add about 50 cc. of water
and sufficient ether to make a layer i cm. deep. Add 5 drops of iodeosine solu-
tion and run in enough o.oi n-potassium hydroxide solution, shaking vigorously
after each addition, to turn the aqueous layer a permanent pale red.
Calculation. 100 grams (= alkaloids from 10 grams of nux vomica) of the
original 150 grams of ether-chloroform mixture were used. The alkaloids were
dissolved by 10 cc. of o.i n-hydrochloric acid and the volume was brought to
100 cc. The excess of acid in 50 cc. of this solution (=5 grams of nux vomica)
was determined by titration with o.oi n-potassium hydroxide. If strychnine
and brucine are present in nux vomica in equal amount, the average equivalent
weight of the two alkaloids is 364. Therefore 1000 cc. of o.i n-hydrochloric
acid correspond to 36.4 grams of alkaloids.
Example. Suppose that the titration of the excess of acid in 50 cc. of solution
required 15.6 cc. of o.oi n-potassium hydroxide = 1.56 cc. of o.i n-alkali. Then
QUANTITATIVE ESTIMATION OF ALKALOIPS 295
5 ~ 1-56 = 3-44 cc. of o.i n-hydrochloric acid are combined with the alkaloids
in 5 grams of nux vomica. According to the proportion
Cc. o.i n-HCl: Grams alkaloid
looo : 36.4 = 3-44: x(x = 0.12522)
3.44 cc. of o.i n-acid are combined with 0.12522 gram of alkaloid, correspond-
ing to an alkaloid content of 20 X 0.12522 = 2.50 per cent. The German
Pharmacopoeia places this percentage as the minimum for total alkaloids in
nux vomica.
2. In Extract of Nux Vomica. Dissolve i gram of extract in an Erlenmeyer
flask in 5 grams of water and 5 grams of absolute alcohol, and add 50 grams of
ether and 20 grams of chloroform to this solution. Shake vigorously and add 10
cc. of sodium carbonate solution (1:3). Let the mixture stand and agitate at
frequent intervals for an hour. * Then pass 50 grams of the clear ether-chloro-
form solution through a dry filter kept well covered, and receive the filtrate in a
flask. Distil half the solvent and pour the remainder into a separating funnel.
Wash the flask three times with 5 cc. portions of a mixture of 3 parts of ether and
i part of chloroform. Thoroughly extract the total ether-chloroform solution
with 50 cc. of o.oi n-hydrochloric acid. When the liquids have separated
clear, if necessary, after addition of enough ether to bring the ether-chloro-
form solution to the surface, pass the acid solution through a small filter mois-
tened with water and receive the nitrate in a 200 cc. flask. Make three extrac-
tions of the ether-chloroform solution with 10 cc. portions of water, and pass
these washings through the same filter. Finally, wash the filter with water and
bring the entire solution to 100 cc. Add enough ether to make a layer i cm.
thick and 5 drops of iodeosine solution. Run in o.oi n-potassium hydroxide
solution, shaking vigorously after each addition, until the aqueous solution is
pale red.
Calculation. Only 50 grams, or two-thirds of the original 75 grams of alcohol-
ether-chloroform mixture, were used. The alkaloids in 0.666 gram of nux
vomica extract were in this volume of solvent. Alkaloids were dissolved in 50
cc. of o.oi n-hydrochloric acid and excess of acid determined by titration with
o.oi n-potassium hydroxide solution.
Example. Suppose that 18 cc. of o.oi n-potassium hydroxide solution were
used in this titration. Then 50 - 18 = 32 cc. of o.oi n-acid were combined
with the alkaloids in 0.666 gram of extract. According to the proportion
Cc. o.oi n-HCl : Grams alkaloids
1000 : 3.64 = 32 : x (x = 0.11648)
0.666 gram of extract contains 0.11648 gram of alkaloids, corresponding to 17.47
per cent. The German Pharmacopoeia places this percentage as the minimum
for total alkaloids in extract of nux vomica.
3. In Tincture of Nux Vomica. Evaporate 50 grams of tincture of nux vomica
in a
. .
weighed dish to 10 grams. Wash this residue into an Erlenmeyer flask and
inse with 5 grams of absolute alcohol. Add 50 grams of ether and 20 grams of
chloroform and shake vigorously. Then add 10 cc. of sodium carbonate solutioi
(1:3) which has been previously employed in rinsing the dish used in evaporatinj
the tincture. Let this mixture stand an hour, shaking vigorously at fre-
296 DETECTION OF POISONS
quent intervals. Filter 50 grams of the clear ether-chloroform solution. To ex-
tract alkaloids, use 40 cc. of o.oi n-hydrochloric acid. In other respects, the
estimation of alkaloids is the same as described for extract of nux vomica.
Calculation. This is the same as that given for extract of nux vomica. Only
two-thirds (= 33.3 grams) of the original weight of nux vomica tincture were
used. The alkaloids were dissolved in 40 cc. of o.oi n-hydrochloric acid and ex-
cess of acid was determined by titration with o.oi n-potassium hydroxide
solution. If 17 cc. of the latter solution were used, then 40 17 = 23 cc. of
o.oi n-hydrochloric acid were combined with the alkaloids in 33.3 grams of the
tincture. According to the proportion
Cc. o.oi n-HCl : Grams alkaloids
1000 : 3.64 = 23 : x (x = 0.08372)
this weight of tincture contains 0.08372 gram of alkaloids, corresponding to 2.51
per cent. The German Pharmacopoeia places this percentage as the minimum
for total alkaloids in tincture of nux vomica. Brucine and strychnine are as-
sumed to be present in equal quantity.
Estimation of Alkaloids in Nux Vomica and Its Preparations by Means
of Picrolonic Acid
(H. Matthes and 0. Rammstedt)
Nux vomica upon the average contains strychnine and
brucine in equal quantity combined with tannic acid. Alkaline
hydroxide or carbonate solutions liberate the alkaloids from
their salts. The free bases are then extracted with an ether-
chloroform mixture. The solvent is reduced to smaller volume
by evaporation or distillation and the alkaloids are precipitated
with picrolonic acid.
Strychnine picrolonate, CnH^NaC^.CioHsN^s (Mol. Wt.
598) melts with decomposition at 286.
Brucine picrolonate, C23H 26 N2O4.CioH 8 N 4 O 6 (Mol. Wt. 658)
melts with decomposition at 277.
i. Extract of Nux Vomica. Dissolve i gram of extract in 5
grams of absolute alcohol and 5 grams of water. Shake well
with 50 grams of ether and 20 grams of chloroform. Add 10 cc.
of sodium carbonate solution (i : 2) and again shake thoroughly
for 10 minutes. Let the mixture stand at rest for 20 minutes.
Pass 50 grams of the ether-chloroform mixture through a dry,
double, creased filter and evaporate half the solvent in a beaker.
Add about 5 cc. of o.i n-alcoholic picrolonic acid to the warm
QUANTITATIVE ESTIMATION OF ALKALOIDS 297
solution. A yellow crystalline precipitate of strychnine and
brucine pkrolonates soon appears. After 24 hours collect the
mixed picrolonates in a weighed Gooch crucible. Wash
excess of picrolonic acid from the precipitate with 2 cc. of an
alcohol-ether mixture (i 13), dry 30 minutes at 110, cool in
desiccator and weigh.
Calculation. Use the mean molecular weight of brucine and strychnine
picrolonates (= 628) and also the mean molecular weight of brucine and" strych-
nine (= 364). The proportion is
Grams picrolonate Grams strychnine Wt. of pre-
mixture : and brucine = cipitate : x.
628 : 364
Since the quotient 364:628 = 0.5798, the weight of mixed alkaloids is obtained
by multiplying the weight of the picrolonate precipitate by this quantity. This
precipitate represents total alkaloids in 0.666 gram of extract of nux vomica,
for only two-thirds (= 50 grams) of the original 75 grams (5 grams of alcohol +
50 grams of ether + 20 grams of chloroform) of solvent, containing the alkaloids
in i gram of extract of nux vomica, were used.
2. Tincture of Nux Vomica. Evaporate 50 grams of tincture
in an Erlenmeyer flask to 10 cc. Cool and shake well with 5 cc.
of absolute alcohol, 50 grams of ether and 20 grams of chloro-
form. Add 10 cc. of sodium carbonate solution (i : 2) and
shake again for 10 minutes. After 20 minutes pass 50 grams
(= two-thirds of the original mixture) of the ether-chloroform
mixture through a double, creased filter. Evaporate half the
solvent in a beaker and add 5 cc. of o.i n-alcoholic picrolonic
acid to the warm residue. Treat the picrolonate precipitate as
described above.
3. Nux Vomica. Exhaust 15 grams of powdered nux vomica,
previously dried at 100, by thoroughly agitating with 100 grams
of ether and 50 grams of chloroform. Then add 10 cc. of a
mixture of 2 parts of 15 per cent, sodium hydroxide solution and
i part of water and shake again for 10 minutes. Add an ad-
ditional 15 cc. of water, or enough to cause the powder to gather
into balls after vigorous agitation and leave the supernatant
ether-chloroform mixture clear. After 30 minutes pass the
clear ether-chloroform solution through a dry, double, creased
filter. Evaporate 50 cc. of the filtrate in a beaker nearly to
298 DETECTION OF POISONS
dryness and add a second 50 cc. portion of nitrate to the
residue, bringing everything into solution (= alkaloids from 10
grams of nux vomica). Add 5 cc. of o.i n-alcoholic picrolonic
acid and treat the precipitate as previously described.
Estimation of Strychnine in Mixtures of Nux Vomica Alkaloids
(Gordin's 1 Modification of Keller's Method)
Strong nitric acid, gently heated with a solution of strych-
nine and brucine in 3 per cent, sulphuric acid, is without action
upon the former alkaloid. But brucine is converted into non-
basic substances not extracted by chloroform from an alkaline
solution.
Procedure. Dissolve the mixed alkaloids (0.2-0.3 gram)
upon the water-bath in 15 cc. of 3 per cent, sulphuric acid and
add 3 cc. of a diluted nitric acid (equal parts of 68-69 per cent,
acid (sp. gr. 1.42) and water) to the cold solution. Pour the
mixture into a separating funnel after exactly 10 minutes and
add sodium hydroxide solution in excess. Extract strychnine
3-4 times with chloroform. Pass the chloroform solution
through a double filter into a small weighed flask, wash the
filter with a little chloroform, add 2 cc. of pure amyl alcohol and
distil to dryness upon the water-bath. Remove the last traces
of liquid, consisting mainly of amyl alcohol, by forcing a current
of air through the flask warmed upon the water-bath. Finally
dry the residue 2 hours at 135-140 and weigh when cold. In
this way a very pure, white strychnine free from brucine is
obtained.
Notes. According to Gordin, ammonia cannot be substituted for sodium
hydroxide, for it gives colored strychnine. Amyl alcohol is added to the chloro-
form solution to -pre vent strychnine crystals from being carried by decrepitation
into the condenser during distillation.
Estimation of Theobromine and Caffeine in Cacao and Chocolate 2
Cacao and its preparations contain only very little caffeine
which is usually determined with theobromine.
1 Archiv der Pharmazie 240, 643 (1902).
2 H. Beckurts, Archiv der Pharmazie, 244, 486 (1906).
QUANTITATIVE ESTIMATION OF ALKALOIDS 299
Boil 6 grams of powdered cacao, or 12 grams of chocolate, for
30 minutes under a reflux condenser in a weighed liter flask with
200 grams of a mixture of 197 grams of water and 3 grams of
dilute sulphuric acid. Then add 400 grams of water and 8
grams of finely powdered magnesia and boil for an hour longer.
When the mixture is cold, add exactly enough water by weight
to replace what has been lost by evaporation. After the mix-
ture has settled, filter 500 grams of solution (=5 grams of cacao
or 10 grams of chocolate) and evaporate the filtrate to dryness
in a dish either by itself or with some quartz sand. Triturate
the residue and exhaust in a Soxhlet tube with chloroform.
In case of evaporation without quartz sand, rub the residue
with a few drops of water, transfer to a separating funnel with
10 cc. of water and extract 8 times with 50 cc. portions of hot
chloroform. Pass the chloroform extract through a dry filter
into a tared flask, distil the chloroform and dry the residue
(= theobromine and caffeine) at 100 to constant weight.
Carbon tetrachloride is used to separate theobromine from
caffeine, the latter alkaloid alone being soluble at room tem-
perature. Let the weighed residue from chloroform stand for i
hour with 100 grams of carbon tetrachloride at room tempera-
ture. Shake occasionally and then filter. Distil carbon
tetrachloride and extract the residue repeatedly with water.
Evaporate the aqueous solution in a weighed dish and dry the
residue (= caffeine) at 100 to constant weight.
Repeatedly extract the theobromine, insoluble in carbon
tetrachloride, and also the filter paper with water. Filter,
evaporate the total filtrate and weigh the residue (= theo-
bromine) dried at 100.
Notes. In the method described above, H. Beckurts and Fromme eliminate
injurious effects due to concentration by boiling with dilute sulphuric acid.
Xanthine bases are set free from combination with organic acid and recombined
with sulphuric acid. Magnesia sets these bases free from their sulphates and
at the same time holds back coloring matter and fat, thus eliminating these
impurities.
Theobromine, 3,7-dimethyl-xanthine, C 7 H 8 N 4 O2, is a white powder consisting
of microscopic needles having a bitter taste. It dissolves in 3282 parts of cold
and 148 parts of boiling water; in 422 parts of boiling absolute alcohol; and in
105 parts of boiling chloroform. Theobromine solutions are neutral. This
300 DETECTION OF POISONS
alkaloid acts both as an acid and as a base and therefore is soluble in both acid
and alkaline solutions. The salts with acids crystallize well but are not very
stable. These theobromine salts are partially decomposed into theobromine
and acid in presence of much water, or, if the given acid is volatile, by heating at
100. Theobromine is isomeric with theophylline, or i,3-dimethyl-xanthine,
and paraxan thine, or i,7-dimethyl-xan thine:
HN CO (i)CH,.N CO (i)CH,.N CO
| | /CH,( 7 ) | | H || /CH,( 7 )
OC C N< OC C N x OC C N<
I II >CH | || >CH | || >CH
( 3 )CH 3 .N C N^ ( 3 )CH 3 .N C N^ HN = C N^
Theobromine Theophylline Paraxanthine
Theophylline occurs in tea leaves and paraxanthine has been isolated from human
urine. The latter is therefore called urotheobromine.
Estimation of Alkaloids in Leaves of Atropa Belladonna, Hyoscyamus
Niger and Datura Strammonium
(E. Schmidt's Modification of Keller's Method 1 )
Shake vigorously 10 grams of finely powdered leaves, dried to constant weight
over quicklime, in an Erlenmeyer flask with 90 grams of ether and 30 grams of
chloroform. Add 10 cc. of 10 per cent, sodium hydroxide solution and shake
vigorously and often for 3 hours. Then add 10 cc. of water, or enough to cause
the powder to gather into balls when thoroughly shaken. After i hour pass 60
grams of the ether-chloroform extract (= 5 grams of leaves) through a dry
filter kept well covered. Distil 60 cc. of this filtrate to half its volume to remove
ammonia, and transfer the deep green solution to a separating funnel, rinsing
the flask with three 5 cc. portions of ether. Shake the combined extracts well
with 10 cc. of o.oi n-hydrochloric acid. Add enough ether to cause the ether-
chloroform solution to rise to the top, and pass the acid solution through a moist
filter into a 200 cc. glass stoppered flask. Shake the ether-chloroform solution
3 times with 10 cc. .portions of water, pouring these extracts through the same
filter and washing the latter with enough water to bring the total volume to 100
cc. Add enough ether to make a layer i cm. deep and 5 drops of iodeosine solu-
tion. Having determined beforehand the exact relation of acid to alkali, titrate
excess of o.oi n-hydrochloric acid with o.oi n-potassium hydroxide solution.
The calculation is the same as that for extract of belladonna (see page 301).
Notes. Using this method, E. Schmidt obtained 0.4 per cent, of alkaloid in
wild belladonna leaves but only 0.26 per cent, in cultivated leaves. The average
of many determinations gave 0.4 per cent, in strammonium leaves and 0.27-0.28
per cent, in hyoscyamus leaves without stalks. Alkaloids were calculated as
atropine.
Sodium hydroxide solution liberates alkaloids from the acids with which they
are naturally combined in the plant, for example:
(Ci7H 23 NO 3 )2.H 2 S04 2 + 2NaOH = 2 Ci 7 H 23 NO3 + 2H 2 O + Na 2 S0 4 .
1 Apotheker-Zeitung 15, 13.
2 The formula of atropine sulphate used in medicine.
QUANTITATIVE ESTIMATION OF ALKALOIDS 301
Estimation of Alkaloids in Extract of Belladonna
(German Pharmacopoeia)
Dissolve 2 grams of extract of belladonna in an Erlenmeyer flask in 5 grams of
water and 5 grams of absolute alcohol, and add 50 grams of ether and 20 grams of
chloroform to this solution. Shake vigorously and add 10 cc. of sodium car-
bonate solution (i : 3). Let the mixture stand and agitate at frequent intervals
for an hour. Then pass 50 grams of the clear ether-chloroform solution through a
dry filter kept well covered, and receive the nitrate hi a flask. Distil half the
solvent and pour the remainder into a separating funnel. Wash the flask three
times with 5 cc. portions of ether. Thoroughly extract the total ether-chloro-
form solution with 20 cc. of o.oi n-hydrochloric acid. When the liquids have
separated clear, if necessary, after addition of enough ether to bring the ether-
chloroform solution to the surface, pass the acid solution through a small filter
moistened with water and receive the nitrate in a 200 cc. flask. Make three
extractions of the ether-chloroform solution with 10 cc. portions of water, and
pass these washings through the same filter. Finally, wash the filter with water
and bring the entire solution to 100 cc. Add enough ether to make a layer i cm.
thick and 5 drops of iodeosine solution. Run in o.oi n-potassium hydroxide
solution, shaking vigorously after each addition, until the aqueous solution is
pale red.
Calculation. Sodium carbonate like sodium hydroxide liberates the alkaloids
atropine and hyoscyamine, from their salts in belladonna leaves:
(C 1 7H23N0 3 )2.H 2 SO4 + Na 2 CO 3 = 2Ci 7 H 23 N0 3 + Na 2 S04.+ CO 2 + HjO.
The free alkaloids dissolve in the alcohol-ether-chloroform mixture. Fifty grams
of this solution ( = alkaloids from 1.33 grams of extract) are extracted with 20 cc.
of o.oi n-hydrochloric acid, the alkaloids passing into the aqueous solution as
salts of hydrochloric acid (CiyH^NOs.HCl). Excess of acid in this solution is
determined by titration. If 13 cc. of o.oi n-potassium hydroxide solution are
used, then 20 13 = 7 cc. of o.oi n-hydrochloric acid correspond to the alka-
loids in 1.33 grams of extract. The equivalent weight of the two isomeric bases,
atropine and hyoscyamine, being 289, 1000 cc. of o.oi n-hydrochloric acid corre-
spond to 2.89 grams of alkaloids. The proportion
1000 : 2.89 = 7 : x (x = 0.02023)
shows that 1.33 grams of belladonna extract contain 0.02023 gram of alkaloids
corresponding to 1.51 per cent. The German Pharmacopoeia places this per-
centage as the minimum for total alkaloids in extract of belladonna.
Extract of Hyoscyamus
The alkaloids in 2 grams of this extract are determined in the manner described
for extract of belladonna. Use 10 cc. of o.i n-hydrochloric acid instead of 20 cc.
to extract alkaloids. The German Pharmacopoeia requires that not more than
6.5 cc. of o.oi n-potassium hydroxide solution shall be used in titrating the excess
of hydrochloric acid. Therefore 10 - 6.5 = 3-5 cc. of o.oi n-hydrochloric acid
are combined with the alkaloids in 1.3 grams of henbane extract ( =
of the original extract). The proportion
1000 : 2.89 = 3.5 : x (x = o.oion)
302 DETECTION OF POISONS
shows that 1.33 grams of extract contain o.oioi gram of alkaloid, corresponding
to 0.76 per cent. This percentage is placed as the minimum for total alkaloids
in henbane extract.
Assaying Officinal Extracts
(E. Merck 1 )
With a view to obviating as many sources of error as possible, E. Merck has
proposed the following procedures :
Extract of Belladonna. Dissolve 4 grams of extract in 6 cc. of water and wash
the solution into a separating funnel with an additional 10 cc. Add 100 cc. of
ether, shaking well, then 10 cc. of sodium carbonate solution (1:3) and shake at
once for 5 minutes. Stopper the funnel and let the mixture stand for 20 minutes.
Then pass the ether layer through a dry filter (10 cm. in diameter) into a glass-
stoppered flask. To lessen evaporation of ether as much as possible, cover the
funnel with a glass plate. If an emulsion keeps the ether from separating well,
add a few grams of powdered tragacanth at the end of the time stated above.
Shake until the tragacanth gathers into balls in the aqueous layer. After 15
minutes decant 75 cc. of the ether layer. To check results by making more than
one assay, use 25 cc. of the ether solution (= i gram of extract). Test a clean
glass-stoppered flask to make sure that it does not give up alkali to the water.
Then introduce into such a flask 50-60 cc. of water, 5 drops of iodeosine solution
and 20 cc. of ether. Shake and add o.oi n-hydrochloric acid until the aqueous
layer just becomes colorless upon shaking. This procedure obviates a special
determination of the alkalinity of the water, since the resulting mixture is brought
to the neutral point. Now add 25 cc. of the ether solution of the alkaloid and
titrate until there is no color. Multiply the number of cc. of o.oi n-hydrochloric
acid used by 0.00289. 2 The product is the quantity of alkaloid, calculated as
atropine, in i gram of belladonna extract. Upon the average this preparation
contains 1.8 per cent, of alkaloid.
Extract of Cinchona. Haematoxylin is frequently an unsatisfactory indicator
in the titration of cinchona alkaloids, because the color change is slow enough
to make it difficult to fix the end-point exactly. Therefore E. Merck makes a
gravimetric and volumetric determination at the same time by the following
method:
Dissolve 3 grams of aqueous cinchona extract in 10 cc. of water in a porcelain
dish. Pour the solution into a 250 cc. shaking flask, rinsing it in with 10 cc.
of water. Add 150 cc. of ether and 10 cc. of sodium carbonate solution (1:3)
to this mixture and shake vigorously for TO minutes. Cork the flask and let the
mixture stand at rest for 30 minutes. This extract frequently forms an emulsion.
In that case add a few grams of tragacanth powder which has no effect upon the
result. Pour the ether solution of cinchona alkaloids as rapidly as possible
through a dry creased filter. Use 30 cc. ( = i gram of cinchona extract) for each
determination. Distil the solvent from the 50 cc. in a weighed 100 cc. flask and
1 Zeitschrift fur analytische Chemie 41, 584 (1902) and also Merck's Bericht
iiber das Jahr; 1900.
2 289 = the equivalent weight of the two isomeric bases, atropine and hyoscy-
amine, CnH^sNOs.
QUANTITATIVE ESTIMATION OF ALKALOIDS 303
dry the residue in an air bath at 100-110 to constant weight. The alkaloids
obtained are nearly colorless or faintly yellow. Having ascertained the weight
of the alkaloids, proceed with the titration. Dissolve the residue in the flask in
10 cc. of alcohol, adding 50 cc. of water, which partially precipitates the alkaloids,
and then alcoholic haematoxylin solution. 1 Run in o.i n-hydrochloric acid until
the alkaloids again dissolve and the red color of the solution passes through
reddish yellow into a pure yellow. The mean equivalent weight of the cinchona
alkaloids is 309. Therefore i cc. of o.i n-hydrochloric acid = 0.0309 gram of
alkaloid. Upon the average, officinal aqueous extract of cinchona contains 9
per cent, of alkaloid.
Extract of Nux Vomica. Dissolve o.i gram of this extract in a flask in 5
grams of absolute alcohol and 10 grams of water. Add 95 grams of ether and
shake well. Then add 10 cc. of sodium carbonate solution (1:3) and shake
vigorously at once for about 10 minutes. After 15 minutes pour the ether solu-
tion as rapidly as possible through a creased filter. Weigh in a flask 50 grams of
this solution (= 0.05 gram of the original extract), having previously placed in
this flask a neutral mixture of 50 cc. of water, 20 cc. of ether and 5 drops of iod-
eosine solution. Add 20 cc. of o.oi n-hydrochloric acid and titrate with o.or
n-potassium hydroxide solution until the aqueous layer is just red.
Calculation. Since strychnine and brucine are present in nux vomica in nearly
equal parts, the mean equivalent weight of such a mixture of bases (334 + 394) : 2
= 364. Hence 0.00364 gram of the mixed alkaloids neutralizes i cc. of o.oi
n-hydrochloric acid. The officinal extract of nux vomica contains 18 per cent,
of alkaloid.
1 E. Merck advises keeping on hand an alcoholic solution of haematoxylin,
because a freshly prepared solution usually gives a blue-violet instead of a red
color change.
CHAPTER VII
DETECTION OF CARBON MONOXIDE BLOOD, BLOOD STAINS
AND HUMAN BLOOD
i. Carbon Monoxide Blood
Carbon monoxide (CO) has a direct toxic action upon the
blood. This gas passed into blood displaces loosely bound
oxygen from oxyhaemoglobin forming the more stable carboxy-
hsemoglobin. The latter compound is cherry-red, not dichroic
and entirely resistant to putrefaction if air is excluded. In
carbon monoxide poisoning the cherry-red color of the blood
is usually noticed at once.
Detection of Carbon Monoxide Blood
1. Boiling Test. Blood containing carbon monoxide gives a
brick -red coagulum, if boiled or warmed upon the water-bath.
Ordinary blood gives a grayish brown or brownish black
precipitate.
2. Sodium Hydroxide Test. Carbon monoxide blood shaken
with 1-2 volumes of sodium hydroxide solution (sp. gr. 1.3
= 26.8 per cent.) remains red and in a thin layer is the color of
red lead or vermilion. Normal blood similarly treated is al-
most black and in a thin coating upon a porcelain plate is dark
greenish brown. A procedure recommended consists in diluting
the blood with 6-10 times its volume of water and using about 5
drops of sodium hydroxide solution to 10 cc. of diluted blood.
Even gentle warming with sodium hydroxide solution (10 per
cent. NaOH) does not alter the red color of this carboxyhsemo-
globin solution, whereas a solution of normal human blood
becomes greenish to dark brown.
3. Basic Lead Acetate Test. Mix 4-5 volumes of basic
lead acetate solution in a test-tube with diluted or undiluted
carbon monoxide blood and shake vigorously for a minute.
304
DETECTION OF CARBON MONOXIDE BLOOD 305
Such blood remains bright red but normal blood is first brownish
and then chocolate to greenish brown.
4. Potassium Ferrocyanide Test. Mix undiluted blood (15
cc.) with an equal volume of 20 per cent, potassium ferrocyanide
solution and 2 cc. of diluted acetic acid. 1 Shake the mixture
gently and a coagulum will gradually form. That from normal
blood is dark brown but from blood containing carbon monoxide
bright red. This difference disappears slowly but not entirely
for weeks.
5. Tannin Test. Mix an aqueous blood solution 2 with 3
times its volume of i per cent, tannin solution and shake thor-
oughly. A difference in color between normal and carbon
monoxide blood can be recognized after several hours, most
distinctly after 24 hours. Normal blood is gray but carbon
monoxide blood is crimson-red. This difference is apparent
even after several months. Ten per cent, of carboxyhaemo-
globin can be detected in blood by tests 4 and 5.
6. Copper Sulphate Test. A drop of saturated copper sul-
phate solution added to 2 cc. of carbon monoxide blood mixed
with the same volume of water gives a brick-red precipitate.
The deposit from normal blood is greenish brown. In all these
precipitation tests (4, 5 and 6) the less easily decomposed
carbon monoxide blood remains bright red but the more easily
decomposed normal blood in presence of the precipitants used
and others is off color or dark.
7. Ammonium Sulphide Test. Mix 0.2 cc. of ammonium
sulphide solution and 0.2-0.3 cc. of 30 per cent, acetic acid with
10 cc. of 2 per cent, aqueous blood solution. Carbon monoxide
blood gives a fine rose color but normal blood is greenish gray.
The former within 24 hours gives a red flocculent precipitate.
8. Palladous Chloride Test. Carbon monoxide precipitates
black metallic palladium from a neutral aqueous palladous
chloride solution:
CO + H 2 O + PdCl 2 = C0 2 + 2HC1 + Pd.
1 Mix i volume of glacial acetic acid with 2 volumes of water. This acid con-
tains about 30 per cent, acetic acid.
2 Use i part of blood to 4 parts of water.
306
DETECTION OF POISONS
Mix a few drops of potassium hydroxide solution with the blood
and warm gently upon the water-bath. By means of a suction
pump draw through the solution air that has been washed until
pure. Pass the gas evolved first through lead acetate solution
to remove possible hydrogen sulphide, then through sulphuric
acid to absorb ammonia and finally through a neutral light red
palladous chloride solution (i: 500).
9. Spectroscopic Examination. The detection of carboxy-
haemoglobin with the spectroscope is comparatively easy. The
Oxyhaemoglobin.
Haemoglobin.
Methaemoglobin.
Carboxyhaemoglobin.
Hsematoporphyrin, very
dilute, acid.
Haematoporphyrin, not so
dilute, alkaline.
FIG. 24. Absorption-Spectra.
two absorption-bands of this compound are quite similar to
those of Oxyhaemoglobin but they lie somewhat nearer together
and more toward the violet. The main difference, however,
between the absorption-bands of these compounds is that those
of carboxyhsemoglobin are not extinguished by reducing agents.
To prepare the blood solution for spectroscopic examination,
dilute 1-1.5 parts of blood with 100 parts of water and make the
DETECTION OF CARBON MONOXIDE BLOOD 307
observations through a layer i cm. thick. To reduce i per
cent, blood solution, mix thoroughly with a few drops of am-
monium sulphide solution and add 4-6 drops more of the same
reagent as a surface layer to exclude air. Reduction begins in
about 6-8 minutes. A solution of tartaric acid and ferrous
sulphate in presence of an excess of ammonium hydroxide
solution will also reduce oxyhaemoglobin.
Oxyhaemoglobin under these conditions is changed to reduced
hemoglobin. The two absorption-bands characteristic of the
former disappear and a broad diffuse absorption-band occupies
the previous bright space between the two bands. The spec-
trum of carboxyhaemoglobin remains unchanged only when 27
per cent, at least of the haemoglobin is saturated with carbon
monoxide. If allowed to stand in an open vessel, blood will lose
carbon monoxide within 8 days. But carbon monoxide blood
sealed in glass tubes is said to keep for years. Carbon monoxide
has been detected in blood of a cadaver after 18 months.
Tollens 1 recommends adding some formaldehyde to the blood solution. This
reagent has not the slightest effect upon the two oxyhaemoglobin bands. Warm-
ing the mixture very gently with ammonium sulphide solution develops a third
and nearly as distinct black band almost midway between the original bands
which gradually disappear. Finally only this band will remain. This is a far
more satisfactory test than that given by the indefinite band of blood alone.
If the solution is cooled and agitated with air, this third band will disappear and
the two original oxyhsemoglobin bands will return.
If carbon monoxide is present, formaldehyde does not have this action.
2. Detection of Blood Stains
The detection of blood in dry stains upon fabrics, wood,
knives, weapons, etc., 2 is more certain and less open to question,
if haemin crystals (Teichmann's blood crystals) are prepared
from the blood pigment. If haemin crystals are obtained, the
stain in question may be regarded with certainty as due to blood.
Fresh blood when dry is bright red and has a smooth surface.
1 Berichte der Deutschen chemischen Gesellschaft 34, 1426 (1901).
2 Blood mixed with iron oxide as, for example, blood upon rusty knives and
weapons usually fails to give haemin crystals.
308 DETECTION OF POISONS
Flakes of such blood scraped from any material are garnet-red
by transmitted light. A solution of fresh blood stains in potas-
sium or sodium hydroxide is dichroic, being red by transmitted
and green by reflected light. Later dried blood becomes
brownish red or dark brown. These color changes are due to
conversion of oxyhaemoglobin into methaemoglobin and then
into haematin. The first two substances are soluble in water
but the last is not. But haematin is soluble in alkalies and in
alcohol containing sulphuric acid. This change of the blood
pigment depends not only upon the age of the stain but really
upon the action of air (oxygen), light,, heat and moisture upon
the blood before it is dry. If the blood is in a thin layer,
haemoglobin will sometimes change into methaemoglobin even
in 3-10 days. Boiling water causes immediate insolubility.
The action is also very rapid in direct sunlight. Washing in
alkaline solutions (boiling solutions of potassium or sodium
soap, sodium carbonate solution, ammonia and sewage) also
causes rapid decomposition. But acids, nitric and hydro-
chloric, as well as putrefaction, act more slowly, giving the blood
a laked appearance and even making it clear and colorless. If,
however, the blood has once dried, these injurious agencies,
even putrefaction, act less easily.
Preparation of Haemin Crystals. Prepare a cold aqueous
extract of the stain as free as possible from fibers and evaporate
the solution upon a watch-glass away from dust. Add a trace
of sodium chloride 1 to the residue, also 8-10 drops of glacial
acetic acid, and stir with a glass rod. Heat just for an instant
over a small flame, then evaporate the solution gradually
upon a moderately warm water-bath and examine the residue
with a microscope magnifying 300-500 times. If haemin
1 Strzyzowski (Chemisches Centralblatt, 1897, I, 295) advises using sodium
iodide instead of sodium chloride. Place a small particle of material suspected
of containing blood upon a glass slide and add a drop of sodium iodide solution
(i : 500). Evaporate and cover with a cover-glass. Heat for 3-6 seconds
with concentrated acetic acid which is allowed to run under the cover-glass. The
test with this modification is said to be more delicate, owing to the darker color
of the hsematin hydriodide crystals. The crystals are usually obtained in less
time and with as small a quantity as 0.000025 gram of fresh blood. Tr.
DETECTION OF CARBON MONOXIDE BLOOD 309
crystals fail to appear, repeat the evaporation several times,
using in each instance 8-10 drops of glacial acetic acid, and
examine the residue each time under the microscope. Hsemin
crystals are brownish red to dark brown and form rhombic scales
which frequently lie crossed (Fig. 25). Usually glacial acetic
acid is the only solvent that will
extract the pigment from old blood
stains. Brucke heats the stains or
scrapings to boiling in a test-tube
with 10-20 drops of glacial acetic
acid. The decanted or filtered
solution, after addition of a trace
of sodium chloride, is evaporated
upon a watch-glass to dryness at
40-80 and the residue is examined FlG aSm ^^ Crystals .
under the microscope. By this
method it is immaterial whether the blood has coagulated or not.
Cold water is without effect upon blood stains, if they have
previously been treated with hot water. Protein substances in
the blood are thus coagulated and rendered insoluble. In
such a case treat the stain with water containing a few drops of
sodium hydroxide solution. If the stains are upon wool, use
very dilute sodium hydroxide solution since alkalies dissolve
wool. Water containing ammonium hydroxide will extract
stains and this alkali does not act upon wool. Use the alkaline
aqueous extract to prepare hsemin crystals. Evaporate the
solution to dryness in a watch-glass upon the water-bath and
mix the residue intimately with 8-10 drops of glacial acetic acid.
Add a trace of sodium chloride and again evaporate. Sometimes
it is advisable, after acidifying the extract of the stain with
acetic acid, to add tannic acid, or zinc acetate, and prepare
Teichmann's crystals from the precipitate.
Occasionally it is necessary to extract suspected stains with
hot alcohol containing sulphuric acid. Haematin formed from
the blood pigment dissolves. If this compound is present, the
solution has a brown color. Excess of sodium hydroxide solu-
tion will produce the dichroism characteristic of an alkaline
310 DETECTION OF POISONS
haematin solution, namely, red by transmitted and green by
reflected light. Obviously, haematin should be identified by
the spectroscope both in acid and alkaline solution.
Blood mixed with iron oxide (blood upon rusty weapons)
usually fails to give haemin crystals but the extract with dilute
sodium hydroxide solution frequently shows the dichroism of
hasmatin solution. Since iron oxide or rust forms an insoluble
compound with haematin, warm such stains for some time upon
the water-bath with sodium hydroxide solution to dissolve any
haematin present.
Haematin. Warming an aqueous blood solution to about 70 decomposes the
blood pigment oxyhaemoglobin into a protein substance called globin and haema-
tin, a pigment containing iron. Acids, alkalies and several metallic salts decom-
pose oxyhaemoglobin in the same way. If this decomposition takes place in the
absence of oxygen, another pigment appears. Hoppe-Seyler gave the latter the
name hsemochromogen and other experimenters have called it "reduced haema-
tin." Oxygen and consequently air rapidly oxidizes this pigment to haematin.
On the other hand reducing agents like ammonium sulphide convert haematin
into haemochromogen. Different formulas are given for haematin. W. Kiister
and others now give it the formula C34H34N 4 FeO5. Haematin is amorphous and
has a dark brown or blue-black color. In water, dilute acids, alcohol, ether and
chloroform it is insoluble but soluble in alcohol or ether containing acid. In
even very dilute solutions of caustic alkalies it is freely soluble. Alkaline haema-
tin solutions are dichroic. In rather thick layers the color appears red by trans-
mitted light and greenish in thin layers. Acid solutions are always brown.
Alkaline haematin solutions are precipitated by calcium or barium hydroxide
solution.
Haemin is the hydrochloric ester of haematin. Very prob-
ably haemin has the empirical formula Cs-iHas^FeC^Cl.
Note. If the blood stain is perfectly fresh, it may be recog-
nized by observing blood-corpuscles with the microscope.
Human blood can be differentiated from animal blood by
comparing blood-corpuscles with those of animal blood as to
size, only when the corpuscles are still intact.
Spectroscopic Detection of Blood
If the extract of a blood stain with cold water is already
brown, a third fainter and narrower band will appear in ad-
dition to the two oxyhaemoglobin bands. This lies in the orange
between C and D and is the methaemoglobin band. Cold
DETECTION OF CARBON MONOXIDE BLOOD 311
water will dissolve most of the methaemoglobin from fresh dried
blood stains.
Acetic acid will discharge these two bands, if the oxyhaemo-
globin solution is not too dilute. At the same time the solution
will become mahogany-brown from formation of haematin in
acid solution. This solution has a characteristic spectrum,
namely, four absorption-bands in the yellow and green. If
excess of ammonium hydroxide is added to this solution, the
alkaline solution contains haematin, recognizable by a broad
faint absorption-band lying between the red and yellow. A
few drops of ammonium sulphide solution will extinguish this
band and bring out two broad bands, namely, one in the green
and the other in the light blue. These bands lie farther to the
right than do those of oxyhaemoglobin and are of about the
same width. This is the spectrum of reduced haematin (haemo-
chromogen). All these spectroscopic tests are very charac-
teristic, especially the spectra of oxyhaemoglobin, haemoglobin
and, in the case of old blood stains, that of reduced haematin.
Up to the present time no red solution has been found which,
upon abstraction and addition of oxygen, will give the same
spectroscopic phenomena as blood.
When the quantity of blood is very small, 01 when the blood
pigment has undergone further decomposition, so that the
bands of oxyhaemoglobin are no longer visible, it is advisable
to extract the stains for several hours with concentrated potas-
sium cyanide solution. Blood will give a light red or yellowish
brown solution containing the cyano-compound of haematin.
The spectrum of haematin in alkaline solution will appear as a
broad, faint band.
The investigations of Kratter and Hammerl have shown that
charred blood, which no longer responds to any of the other
blood reactions, will still 'give the haematoporphyrin spectrum
upon treatment with concentrated sulphuric acid (E. v. Hof-
mann, Lehrbuch der gerichtlichen Medizin, 1903).
Ammoniacal carmine solution gives two absorption-bands
similar to those of oxyhaemoglobin but they do not change
upon addition of acetic acid or ammonium sulphide. A band
312 DETECTION OF POISONS
given by fuchsine analogous to that of haemoglobin remains
unchanged after agitation with air.
Other Blood Tests
i. Schb'nbein-Van Been Ozone Test. A mixture of ozon-
ized turpentine 1 and alcoholic tincture of guaiac resin, shaken
with a little blood, produce a light blue color. Separated from
the turpentine, the tincture is deep blue. Though very
delicate, this test is not characteristic of blood, for many
inorganic and organic substances under the same conditions
produce "guaiac blue." Nitrous acid, chlorine, bromine and
iodine, chromic and permanganic acids, ferric and cupric salts
produce blue solutions direct with guaiac resin. In examining
blood stains usually it is possible to exclude these substances
beforehand. But other substances like cell contents or haemo-
globin, having the power of transferring ozone, may attach
false significance to the guaiac-blue reaction. Enzymes
(diastases), hydrolytic ferments (enzymes in the narrower
sense), as well as the so-called oxidation feiments (oxidases), are
organic substances of this character. They occur in different
parts of plants, especially in fungi and in seeds. Saliva, ex-
tracts of certain organs, contents of white blood-corpuscles and
pus cells are animal products of similar nature. E. Schaer 2
states that these animal and vegetable substances differ from
hydrogen dioxide in being catalytic in action and carriers of oxy-
gen at the same time. And also that a temperature of 100,
or contact with hydrocyanic acid, completely destroys their
power of transferring oxygen, or at least greatly diminishes it, in
which respect they are essentially different from haemoglobin.
Neither high temperature (100) nor hydrocyanic acid has any
restraining influence upon haemoglobin so far as transference of
oxygen is concerned. Consequently an extract, containing one
of these ferment-like substances but no blood, placed even for a
1 Turpentine always contains ozone, if exposed to light for a long time in a
loosely stoppered bottle.
2 Forschungsberichte iiber Nahrungsmittel, etc., 3, i (1896) and Archiv der
Pharmazie 236, 571 (1898).
DETECTION OF CARBON MONOXIDE BLOOD 313
short time in a hot water-bath, loses the power of giving the
"guaiac blue" test. In absence of blood, the result will also
be negative, if the extract of the suspected stain is treated with
hydrocyanic acid. For these reasons great care is necessary in
interpreting a positive guaiac test given by the extract of a
supposed stain. The guaiac test is certainly very useful as a
delicate preliminary test and in many instances as a check upon
blood. The three forms of the blood pigment entering into
such an examination, namely, haemoglobin, methaemoglobin
and haematin, are alike in the guaiac test, at least qualitatively,
as far as transference of oxygen is concerned. The examination
and extraction of the stain may, therefore, be conducted in
neutral, acid or alkaline solution, depending upon the nature of
the substance, and either hot or cold. Render an alkaline
extract faintly acid with acetic acid before adding guaiac
tincture. In many instances it is advisable to extract the blood
stain with hot alcohol containing sulphuric acid. Treat such an
acid, alcoholic hasmatin solution with guaiac tincture direct.
Addition of water will precipitate the resin with the adherent
blood pigment.
(a) Vitali's Procedure. Extract the stain with water con-
taining carbon dioxide, or old stains with very dilute sodium
hydroxide 1 solution free from nitrite and nitrate. F Iter the
extract and add a little alcoholic guaiac tincture to a portion of
the nitrate after acidification with acetic acid, if necessary.
If the milky liquid is not blue in 15 minutes, interfering oxidiz-
ing agents are absent. Then add a few drops of old turpentine
and shake. The milky liquid will turn blue at once, or in a short
time, if blood pigment is present. Very .gentle wanning upon
the water-bath increases the delicacy of the reaction. Even
putrid blood 2 months old is said to give a positive test.
(6) E. Schaer's Procedure. Blood stains upon linen, though
quite old, dissolve completely when treated for some time with
70 per cent, chloral hydrate solution. Moistening the stains
beforehand with glacial acetic acid aids solution. Also pre-
pare an extract of guaiac resin in 70 per cent, chloral hydrate
1 In this test use sodium hydroxide prepared from metallic sodium.
314 DETECTION OF POISONS
solution. Mix the extract of the stain with an equal volume of
the latter solution. In absence of nitrites, the color of this
mixture is brownish yellow to light brown. If preferred, a
contact test for blood may be made by this method. Add to
the mixture of blood and guaiac Hiinefeld's 1 turpentine solu-
tion, or hydrogen peroxide, as a surface-layer. An intense
blue zone will appear where the two solutions meet. Guaia-
conic acid in guaiac resin produces ' ' guaiac blue. ' ' O . Dobner has
suggested substituting a dilute solution of guaiaconic acid for
guaiac resin. Blood, or blood pigment, behaves like a ferment
and activates the ozonized turpentine or hydrogen peroxide,
either of which by itself will not turn the solution of guaiac
resin blue.
2. Schaer's Aloin Test. The same conditions, producing
"guaiac blue" from guaiaconic acid, give rise to "aloin red"
from aloin. This substance has a stronger coloring power and
lasts longer than "guaiac blue." Use the same solution of blood
in 70-75 per cent, chloral hydrate solution mixed with a weak
chloral hydrate solution of aloin. Add Hiinefeld's hydro-
gen peroxide solution as a surface layer. After some time a
violet-red zone will appear and a red color of equal intensity will
gradually extend throughout the aloin solution. Another
method of making this test consists in first extracting the blood
stain with pure water, acetic acid, chloral hydrate solution or
alkaline salt solution. Neutralize this solution and add dilute
alcoholic aloin solution and hydrogen peroxide. If the sus-
pected stain contains blood pigment, a red color will appear or
once and persist for a long time.
3. Biological Detection of Human Blood 2
Injection of bacteria produces specific, bacteriolytic bodies
and similarly injection of the blood of one animal species into
1 See page 321 for the preparation of this reagent.
2 This subject has been introduced for the sake of completeness. If such an
investigation is for forensic purposes, the chemist will either decline to undertake
it, or conduct the experiment with an associate who has had bacteriological and
pathological experience.
DETECTION OF CARBON MONOXIDE BLOOD 315
an animal of a different species gives rise to specific, hamiolytic
and agglutinating bodies. Rabbit's blood, for example, in-
jected repeatedly into a guinea pig, develops in the serum of
such a guinea pig substances capable of agglutinating and dis-
solving red corpuscles of the rabbit, setting hemoglobin free
and rendering the blood laky. Blood serum from an animal,
into which denbrinated blood, or blood serum from a different
animal species has been injected intravenously, subcutaneously
or intraperitoneally, that is to say, into the peritoneal cavity,
has the peculiar property of causing precipitation only in blood
serum of this particular animal species. Uhlenhuth, 1 Wasser-
mann and Schiitze, 2 and others have made independent experi-
ments of this kind with blood serum to find for forensic purposes
a test, based upon this biological method, which shall differ-
entiate human blood from the blood of every other animal
species. Repeated injection of 10 cc. of defibrinated human
blood, or human blood serum free from cells, into a rabbit, either
intraperitoneally or subcutaneously, yields a serum producing a
heavy, cloudy precipitate in an aqueous solution of human
blood. This coagulin is specific in action, producing a precipi-
tate only in presence of human blood. Wassermann and
Schiitze tested the blood of 23 different animals, among which
were mammals, birds and fishes, and obtained negative results
with blood solutions from these very different animal species.
By use of blood serum it is possible to differentiate even old
human blood, dried for many weeks, from the blood of other
animals.
To demonstrate the use of this method, A. Dieudonne 3
prepares i per cent, blood solutions, placing 2 cc. of the clear
filtered solution in small test-tubes and adding an equal volume
of double physiological salt solution (=1.8 per cent. NaCl).
Then add 6 drops of serum to each portion and place the tubes
in an incubator at 37. The serum of the rabbit, treated with
1 Deutsche medizinische Wochenschrift, 1901, No. 6; und Zeitschrift fUr
Medizinalbeamte, 1903, Heft 5 and 6.
^Berliner klinische Wochenschrift, 1901, No. 7.
3 Miinchener medizinische Wochenschrift, 1901, page 533-
316 DETECTION OF POISONS
human blood serum, added to an aqueous solution of 'human
blood, produced in a few minutes a distinct flocculent precipi-
tate which gradually became more and more marked. As a
check, test also with normal rabbit's serum which will cause no
precipitate in a solution of human blood. Dieudonne found
also that rabbit's serum, obtained after injecting human blood
serum, causes precipitates not only in human blood solutions
but in human urine containing albumin, with an exudate from
human pleura and with peritoneal exudate. But precipitation
in the case of human blood was much more marked than in these
other tests. In his experiments Dieudonne used blood ex-
pressed from the placenta, repeatedly injecting it subcutane-
ously into rabbits in separate doses of 10 cc. and at intervals
of 3-4 days. The animals were bled several days after the last
injection and the blood was kept upon ice.
The antiserum used in detecting blood should above every-
thing else be perfectly clear. To prepare such serum, use a
sterile Berkefeld filter attached to a water pump. The anti-
serum should be active in very dilute solution. Distinct tur-
bidity should appear immediately in a solution diluted i : 1000,
or in 1-2 minutes at latest. Sera must be of this high efficiency
for practical use. Uhlenhuth has shown that the biological
method of detecting blood is specific for human albumin. A
necessary consequence of this fact is that the material should
first of all be shown to be blood. The first question for the
expert to answer in such an investigation must always be:
"Is there any blood at all present?" If the answer is affirma-
tive, the next question is: "Is it human or animal blood?"
Consequently the material should first be examined for blood
stains by van Deen's ozone test, Teichmann's haemin test and
by the spectroscope. If the suspected stains are upon a hard
surface, as a knife, hatchet, gun-barrel, wood, stone, etc., they
should be scraped off for the biological blood test and extracted
for several hours in a test-tube with physiological salt solution
(=0.9 per cent. NaCl). First, filter the extract through paper.
If the filtrate is not clear, next use a Berkefeld filter.
APPENDIX
PREPARATION OF REAGENTS
General Alkaloidal Reagents. A class of reagents, known as
general alkaloidal reagents, added to solutions of most of the
alkaloids or of their salts, produce precipitates characterized by
their color, their amorphous or crystalline appearance and their
insolubility or sparing solubility in water. But these reagents
do not precipitate alkaloids exclusively. Several members of
this class, for example, the chlorides of gold, platinum and
mercury, phospho-molybdic and phospho-tungstic acids, react
similarly with ammonia and many ammonium derivatives. An
explanation of this similarity in behavior is found in the fact
that most of the alkaloids, being secondary or tertiary bases, are
themselves ammonium derivatives. Nearly all the general
alkaloidal reagents also precipitate proteins, albumoses, pep-
tones, creatinine and the nuclein bases, adenine, guanine,
hypoxanthine and xanthine.
The general alkaloidal reagents are especially useful in
detecting the presence, or absence, of alkaloids and other basic
compounds. If there is only a slight residue from the ether
extract of the alkaline solution in the Stas-Otto method, test
first with the general alkaloidal reagents and then, if necessary,
for individual alkaloids. To perform these tests, dissolve
the given residue in very dilute hydrochloric or sulphuric acid,
distribute the filtered solution upon several watch-glasses and
add to each portion a drop of the more sensitive reagents.
If an alkaloid or any other basic substance is present, distinct
precipitates or at least decided cloudiness will appear in all or
in nearly all of the tests.
The most important general alkaloidal reagents are the
following :
Gold Chloride dissolved in water (i : 30) produces white, yel-
low or brown precipitates which are amorphous or crystal-
317
318 DETECTION OF POISONS
line. These precipitates decompose to some extent with
separation of metallic gold.
Platinum Chloride dissolved in water (i : 20) produces yel-
lowish white to yellow precipitates which are usually granular
and crystalline. These precipitates are usually analogous in
composition to ammonium chloroplatinate, (EUN^PtCle-
Mercuric Chloride dissolved in water (i : 2,0) produces white
to yellowish precipitates which are usually amorphous but
gradually become crystalline.
lodo-potassium Iodide, prepared by dissolving 5 parts of
iodine and 10 parts of potassium iodide in 100 parts of water,
produces brown precipitates which are usually flocculent.
Potassium Cadmium Iodide, prepared by dissolving 20 grams
of potassium iodide in 20 cc. of boiling water, adding 10 grams
of cadmium iodide and diluting to 100 cc., produces white or
yellowish precipitates with sulphuric acid solutions of most of
the alkaloids, even when these solutions are very dilute. These
precipitates, at first amorphous but later crystalline, dissolve
in an excess of the reagent and also in alcohol.
Potassium Bismuthous Iodide may be prepared according to
Kraut 1 by dissolving 80 grams of bismuth subnitrate in 200
grams of nitric acid (sp. gr. 1.18 = 30 per cent. HNO 3 ) and pour-
ing this solution into a concentrated solution of 272 grams of
potassium iodide in water. Allow the potassium nitrate to
crystallize and dilute the solution with water to 1000 cc. This
reagent produces orange-red precipitates with sulphuric acid
solutions of many alkaloids. By shaking these precipitates
with sodium hydroxide and carbonate solution, it is often pos-
sible to recover the alkaloids unchanged and sometimes almost
quantitatively.
Potassium Mercuric Iodide, prepared by dissolving 1.35
grams of mercuric chloride and 5 grams of potassium iodide in
100 cc. of water, produces white or yellowish precipitates with
hydrochloric acid solutions of most of the alkaloids. These
precipitates, at first amorphous, gradually become crystalline.
1 Annalen der Chemie und Pharmazie, 210, 310 (1882) und Archiv der Phar-
mazie, 235, 152 (1897).
PREPARATION OF REAGENTS 319
Potassium Zinc Iodide is prepared by dissolving 10 grams of
zinc iodide and 20 grams of potassium iodide in 100 cc. of water.
Phospho-molybdic Acid may be prepared by either of the
following methods :
(a) Saturate sodium carbonate solution with pure molybdic
acid, add i part of crystallized disodium phosphate (Na 2 HPO 4 .-
i2H 2 O) to 5 parts of the acid and evaporate to dryness. Fuse
the residue in a porcelain crucible and dissolve the cold melt in
water. Prepare 10 parts of solution from i part of this residue.
Add enough nitric acid to the filtered solution to produce a
golden yellow color.
(b) If molybdic acid is not at hand, completely precipitate at
40 with excess of sodium phosphate solution the nitric acid
solution of ammonium molybdate used in testing for phos-
phoric acid. Thoroughly wash the yellow precipitate, add
water and dissolve in warm concentrated sodium carbonate
solution. Evaporate this solution to dryness and fuse the resi-
due until ammonia is completely expelled. If there is any
reduction (blue or black color), moisten the residue with nitric
acid and fuse again. Dissolve this residue in hot water and
add nitric acid in large excess. Prepare 10 parts of solution
from i part of residue. The golden yellow solution should be
protected from ammonia vapor.
Phospho-molybdic acid produces yellowish, amorphous pre-
cipitates with sulphuric acid solutions of most of the alkaloids.
After a while these precipitates are frequently greenish or
bluish from reduction of molybdic acid to molybdic oxide.
Phospho-tungstic Acid, prepared by adding a little 20 per
cent, phosphoric acid to an aqueous solution of sodium tungs-
tate, produces precipitates similar to those given by phospho-
molybdic acid.
Tannic Acid is a 5 per cent aqueous solution of tannin.
This reagent produces whitish or yellowish, flocculent precipitates
partially soluble in hydrochloric acid. Alkaloids may be
recovered in part from these precipitates by treating them with
lead or zinc carbonate, evaporating to dryness and extracting
the residue with ether, alcohol or chloroform.
320 DETECTION OF POISONS
Picric Acid is a concentrated aqueous solution of picric acid
which produces yellow crystalline precipitates, or amorphous
precipitates which soon become crystalline.
Picrolonic Acid is used as o.i normal alcoholic solution by
dissolving 26.4 grams of solid picrolonic acid (CioH 8 N 4 05) in a
liter of alcohol. With most of the alkaloids this solution pro-
duces salts called picrolonates which are crystalline, difficultly
soluble and yellow to red in color. Picrolonic acid behaves to-
ward bases like a monobasic acid. 1
B. Other Reagents and Solutions
Erdmann's Reagent. Sulphuric acid containing nitric acid,
prepared by adding to 20 cc. of pure concentrated sulphuric
acid 10 drops of a solution of 6 drops of concentrated nitric acid
in 100 cc. of water.
Froehde's Reagent. A solution of molybdic acid in sulphuric
acid, prepared by dissolving 5 mg. of molybdic acid, or sodium
molybdate', in i cc. of hot, pure concentrated sulphuric acid.
This solution, which should be colorless, does not keep long.
Fehling's Solution. The two following solutions, which
should be kept separate, are used in preparing this reagent:
1. Copper Sulphate Solution. Dissolve 34.64 grams of pure
crystallized copper sulphate (CuSO 4 .5H 2 O) in sufficient water
to make 500 cc.
2. Alkaline Rochelle Salt Solution. Dissolve 173 grams of
Rochelle salt (K.Na^H^Oe^H^O) and 50 grams of sodium
hydroxide in hot water and dilute this solution when cold to
500 cc.
These two solutions, mixed volume for volume, constitute
Fehling's solution which should be prepared just before being
used. Fehling's solution, which has been made up and kept,
should always be tested before being used. The solution should
not be used, if it gives a red precipitate of cuprous oxide when
warmed by itself.
1 L. Knorr, Berichte der Deutschen chemischen Gesellschaft, 30, 914 (1897);
H. Matthes and O. Rammstedt, Zeitschrift fiir analytische Chemie 46, 565 and
Archiv der Pharmazie 245, 112 (1907).
PREPARATION OF REAGENTS 321
Formaldehyde-sulphuric Acid. Add 2-3 drops of aqueous
formaldehyde solution (formalin) to 3 cc. of pure concentrated
sulphuric acid just before using.
Giinzfcurg's Reagent. 1 Dissolve i part of phloroglucinol and
i part of vanilline in 30 parts of alcohol. This reagent is used
to detect free mineral acid, especially hydrochloric acid, but it
does not react with free organic acids.
Hiinef eld's Solution. Add 25 cc. of alcohol, 5 cc. of chloro-
form and 1.5 cc. of glacial acetic acid to 15 cc. of old turpentine
which has been exposed for some time to air and light. The
turpentine used should not produce a blue color with guaiac
tincture direct nor with 15 cc. of 3-5 per cent, hydrogen peroxide
free from acid. This solution is used in the detection of blood.
lodic Acid Solution. Prepare a 10 per cent, aqueous solution
of iodic acid (HI0 3 ).
Magnesia Mixture. Dissolve 1 1 grams of crystallized mag-
nesium chloiide (MgCl 2 .6H 2 0) and 14 grams of ammonium
chloride in 130 cc. of water and add 70 grams ol ammonium
hydroxide solution (sp. gr. 0.96 = 10 per cent, of NH 3 ). This
mixture should be clear. It is used to detect arsenic and
phosphoric acids.
Mandelin's Reagent. Dissolve i part of ammonium meta-
vanadate (H 4 N.VO 3 ) in 200 parts of pure concentrated sulphuric
acid.
Millon's Reagent. Dissolve i part of mercury in i part of
cold fuming nitric acid. Dilute with twice the volume of
water and decant the clear solution after several hours.
Nessler's Reagent. Dissolve separately in the cold 3.5
grams of potassium iodide in 10 cc. of water and 1.7 grams of
mercuric chloride in 30 cc. of water. Add mercuric chloride
solution to potassium iodide solution until there is a permanent
precipitate. Dilute with 20 per cent, sodium hydroxide solu-
tion until the volume is 100 cc. Add mercuric chloride solution,
until there is again a permanent precipitate and let the solution
i It is advisable to prepare this reagent as required. Keep two, separate alco-
holic solutions (i : 15) of phloroglucinol and vanilline and mix volun
as needed. Tr.
21
322 DETECTION OF POISONS
settle. Decant the clear solution and keep in small bottles in
the dark. This reagent improves upon standing.
Mecke's Reagent. 1 Dissolve 0.5 gram of selenious acid in
10 grams of pure concentrated sulphuric acid.
Stannous Chloride Solution. Mix 5 parts of crystallized
stannous chloride with i part of hydrochloric acid and com-
pletely saturate with dry hydrochloric acid gas. Let this
solution settle and filter through asbestos. It is a pale,
yellowish, refractive liquid (sp. gr. at least 1.9). This solution
is used to detect arsenic (Bettendorffs Arsenic Test).
C. The Indicator lodeosine
lodeosme, or erythrosine, Cz<>H.&LiQo, is a tetra-iodo-fluoresceine, formed by
treating fluoresceine with iodine, and has the formula:
/ CeHI 2 (OH)\
CfC 6 HI 2 (OH)/ U
I \C 6 H 4 .CO.O
I I
The commercial preparation usually contains as impurities small quantities of
substances almost insoluble in ether. To obtain a pure product, 2 dissolve com-
mercial iodeosine in aqueous ether and extract iodeosine from the filtered ether
solution by means of dilute sodium hydroxide solution. Strong sodium hydrox-
ide solution, added to this aqueous alkaline solution, precipitates the sodium salt
of iodeosine. Filter, wash with cold alcohol and crystallize from hot alcohol.
Well formed, almost rectangular plates having a green color on the surface are
obtained. Hydrochloric acid precipitates pure iodeosine from the aqueous solu-
tion of the sodium salt. Pure iodeosine dried at 1 20 is markedly lighter than the
commercial preparation. It is almost insoluble in absolute ether, benzene and
chloroform; more easily soluble in acetone, alcohol and aqueous ether. The tone
of the purified pigment dissolved in aqueous alkali is yellower than that of the
crude product. lodeosine is a scarlet crystalline powder which dissolves in alco-
hol with a deep red and in ether with a yellowish red color. lodeosine is said to be
insoluble in water containing a trace of hydrochloric acid. To prepare iodeosine
solution for use as an indicator, dissolve i gram of the pigment in 500 grams of
alcohol.
1 Zeitschrift fiir offentliche Chemie 5, 350 (1899).
2 Fr. Mylius and F. Foerster, Berichte der Deutschen chemischen Gesellschaft
24, 1482 (1891).
INDEX OF NAMES
Abel, R., Biological arsenic test, 244.
Mould for same, 245.
Bischoff, C., Carbolic acid in the body,
27. Oxalic acid in the body, 190.
Potassium chlorate in putrefac-
tion, 197.
Archangelsky, Chloral hydrate estima- Black, Gutzeit estimation of arsenic,
240.
Bloemendal, Normal arsenic, 176.
tion, 41. Acetone on dogs, 55.
Autenrieth, W., Phenacetine test, 76.
Morphine in putrefaction, 137. Blondlot, Phosphorus test, 8.
Destruction of organic matter, Bodlander, Tin poisoning, 181.
151. Copper in organs, 178.
B
v. Babo, Destruction of organic matter,
148. Detection of arsenic, 161.
Barger, Ergotoxine, 209.
Baudin, Cantharidin in Spanish flies,
264.
Baumann, E., Carbolic acid in putre-
faction, 27.
Baumert, Nitric acid detection, 185.
Bazlen, M., Maltol, 251.
Beck, Aconine, 262.
Beckurts, H., Carbolic acid estimated,
31. Arsenic isolated as trichlo-
ride, 233. Theobromine and caf-
feine in cacao and chocolate, 298.
Beissenhirtz, Chromic acid test for
aniline, 45.
Benedikt, R., Compound of carbolic
acid and bromine, 29.
Bernard, Arsenic reagent, 247.
Berthelot, M., Ethyl alcohol test, 50.
Bertrand, Normal arsenic, 175.
Berzelius, Arsenic test, 156.
Borsche, Composition of isopurpuric
acid, 71.
Bougault, J., Arsenic reagent, 247.
Boughton, W. A., Apparatus for Gut-
zeit test, 240.
Brand, J., Maltol, 251.
Brandl, J., Haemolysis by githagin,
224.
Bredemann, Colchicin estimation, 269.
Brucke, Acetic acid for blood stains,
39-
Bruger, P., Composition of picrotoxin,
67.
Brunner, H., Caffeine and theobro-
mine estimation, 272.
Brunner, K., Saponin in beverages,
223.
Biidinger, Chloroform in mucus, 35.
Buttenberg, Biological arsenic test,
244. Mould for same, 245.
Caesar, Estimation of pilocarpine, 286.
Caillot, Piperine in pepper, 288
Carles, J., Quinine estimation, 268.
Bettendorff, Arsenic test, 162. Prepa- Carlson, C. E., Arsenic in organic corn-
ration of reagent, 322.
pounds, 245.
Beuttel, F., Carbolic acid determina- Carr, F. H., Ergotoxine, 209.
tion, 31.
Biggs, Methyl alcohol test, 53.
Cazeneuve, Piperine in pepper, 288.
Cerny, Normal arsenic, 176.
Biginelli, Gas from biological arsenic Chancel, Solubility of carbon disul-
test, 243.
phide, 46.
323
324
INDEX OF NAMES
Chittenden, Arsenic in the brain, 174.
Ciamician, Structure of pseudo-pellet-
ierine, 270.
Ckindi, Solubility of carbon disul-
phide, 46.
Couerbe, Narcotine test, 115.
Dale, H. H., Ergotinine, 210.
DenigSs, Methyl alcohol test, 54.
Cocaine test, 109.
Dieterich, K., Cantharidin in Spanish
flies, 264. Analysis of cola nuts
and extracts, 272.
Dieudonne, A., Biological blood test,
3iS.
Dilthey, A., Veronal preparation, 80.
Dobner, O., Guaiaconic acid in blood
test, 314.
Doepmann, Morphine in putrefaction,
137-
Dragendorff, G., Phosphorus in cada-
vers, 16. Narcotine test, 115.
Detection of cantharidin, 205.
Dusart, Phosphorus test, 8.
Eichwede, Compound of carbolic
acid and bromine, 29.
Elfstrand, Blood agglutinated by
ricin, 229.
Elvers, Phosphorus in cadavers, 16.
Engel, Arsenic reagent, 247.
v. Engelhardt, R., Aniline black in
the organism, 44.
Erdmann, Narcotine test, 115. Nar-
ceine test, 139. Thebaine test,
228. Reagent prepared, 320.
Faust, Morphine in the organism, 137.
Fehling, Reagent prepared, 320.
Fihlene, Nitrobenzene absorption-
band, 42.
Fischer, A., Interference with phos-
phorus test, 8.
Fischer, B., Chloroform estimation, 38.
Distribution of same in cadavers,
38. Distribution of ethyl alcohol
in organism, 49.
Fischer, E., Veronal preparation, 80.
Excretion of same, 81. Recovery
from urine/ 82. Murexide reac-
tion, 85.
Fleury, G., Morphine test, 135. Ni-
tric acid detection, 185.
Fluckiger, F. A., Carbolic acid test, 29.
Foerster, F., Preparation of pure
iodeosine, 322.
Forster, A., Estimation of caffeine in
coffee, 272.
Frankel, A., Estimation of phosphorus
in phosphorated oils, 232.
Frerichs, Alkaloids in ipecac, 277.
Frerichs, G. and H., Toxicological
examination for veronal, 81.
Fresenius, C. R., Phosphorus test, 10.
Destruction of organic matter,
148. Detection of arsenic, 161.
Freund, M., Structure of veratrine al-
kaloids, 95. Narcotine structure,
1 14. Identity of pseudo-narceine
and narceine, 138. Aconine, 262.
Froehde, Test for codeine, 112. For
narcotine, 115. For hydrastine,
118. For apomorphine, 128.
For morphine, 134. For papa-
verine, 217. For saponins, 222.
For solanine and solanidine, 227.
For thebaine, 228. For cephae-
line, 278. Preparation of re-
agent, 320.
Fromme, G., Pilocarpine in jaboran-
dum, 286. Theobromine and caf-
feine in cacao and chocolate, 299.
de Fuentas Tapis, Alkaloids in ipecac,
277.
Fiihner, H., Thalleioquin reaction, 121.
Fujiwara, Chloroform test, 37.
Gadamer, J., Cantharidin structure,
203. Structure of cantharic acid
and acetyl-hydrato-cantharic an-
INDEX OF NAMES
325
hydride, 204. Caffeine in tea,
coffee and cola, 272.
Gair, D., Estimation of strychnine and
quinine together, 258.
Gautier, A., Normal arsenic, 1 74. De-
struction of organic matter, 234.
Girard, Ch., Detection of salicylic acid
in milk, 251.
Goeckel, H., Caffeine, 275.
Goldschmiedt, G., Structure of papa-
verine, 216.
Gordin, H. M., Estimation of alka-
loids, 257. Of strychnine, 298.
Gosio, B., Moulds on arsenical media,
242.
Graf, L., Caffeine in tea, 272.
Grandeau, Veratrine test, 96.
Guareschi, Solubility of quinine sul-
phate, 268.
Guglialmelli, Pyramidone test, 125.
Gunning, Acetone differentiated from
ethyl alcohol, 56.
Giinzburg, Mineral acid test, 182.
Reagent for mineral acid, 321.
Gutzeit, Arsenic test, 163.
Hinsberg, Phenacetine test, 76.
Hirschsohn, Quinine test, 122.
Hodlmoser, Normal arsenic, 176.
Hofmann, A. W., Sensitiveness of
phenylisocyanide test, 36. Ex-
haustive methylation, 117.
v. Hofmann, E., Haematoporphyrin
spectrum, 311.
Hofmeister, Lead in sheep, 177.
Holmes, Isolation of methyl alcohol,
S3-
Hoppe-Seyler, F., Haemochromogen,
310-
Huchard, Toxic action of nicotine, 91.
Hunefeld, Preparation of blood re-
agent, 321.
Hurt, H., Electrolytic estimation of
arsenic, 237. Electrodes for
same, 239. Organic matter in ar-
senic determinations, 239.
Husemann, Morphine test, 133.
Ipsen, Resistance of atropine to putre-
faction, 103.
Halasz, Z., Modification of Blondlot-
Dusart phosphorus test, 13. Or-
ganic phosphorus compounds, 13.
Hammerl, Examination of charred
blood, 311.
Harmsen, Carbon disulphide on li-
poids, 46.
Harrison, E. F., Estimation of quinine
and strychnine. 258.
Helch, H., Pilocarpine test, 219.
Hildebrandt, H., Chlorate in urine,
197.
Hilger, Phosphorus test, n. Reduc-
tion of phosphorous acid to phos-
phine, 14. Apparatus to esti-
mate phosphorus, 15. Sensitive-
ness of Mitscherlich test, 16.
Caffeine in coffee and tea, 271.
Hille, W., Solubility of quinine sul-
phate, 268.
Hinkel, Methyl alcohol test, 54.
Jaffe, Rubazonic acid, 124. Santonin
in dogs, 199.
Jaquet, A., Toxic action of ergotinine
and hydro-ergotinine, 210.
Jaworowski, Chloral hydrate differ-
entiated from chloroform, 39.
Jeserich, Destruction of organic mat-
ter, 151.
JoneScu, D., Antipyrine in the organ-
ism, 123. Alkaloids estimated by
potassium bismuthous iodide, 255.
Jowett, Pilocarpine, 217. Formula of
same, 218.
Juckenack, Caffeine in coffee and tea,
Katz, E., Estimation of caffeine, 272,
276.
Katz, J., Estimation of colchicin, 269.
Caffeine in plant products, 276.
Santonin in wormseed, 290.
326
INDEX OF NAMES
Keller, C. C., Ergotinine, 209. Detec-
tion and estimation of same, 211.
Caffeine in tea, 272. Nicotine
in tobacco, 279. Alkaloids in nux
vomica, 293.
Kiliani, H., Digitalinum verum crys-
tallisatum, 207. Hydrolysis of
digitonin, 207. Test for digi-
toxin, 208. Digitalin, 208. Di-
gitalose, 208. Maltol, 251.
Kippenberger,*C., Extraction of mor-
phine, 130.
Kissling, R., Nicotine in tobacco,
279.
Klason, Arsenic and mercuric chloride
compound in biological test, 243.
Kleist, Excretion of veronal, 81. Re-
covery from urine, 82.
Knocke, Solubility of antimony spot,
160.
Knorr, L., Picrolonic acid, 253, 320.
Kobert, L., Solanine extracted with
isobutyl alcohol, 226.
Kobert, R., Toxic action of hydro-
cyanic acid, 20. Chloroform con-
verted in organism to chloride, 35.
Chloral hydrate in brain, 41.
Physiological action of nitroben-
zene, 43. Urine in aniline poi-
soning, 44. Carbon disulphide on
lipoids, 46. Ethyl alcohol in
blood, 49. Acetone in urine, 55.
Picric acid in urine, 70. Acetani-
lide poisoning, 73. Strychnine in
the body, 97. Cocaine physio-
logical test, 109. Metals and red
blood corpuscles, 173. Toxicity
of uranium, 180. Sulphuric acid
poisoning, 187. Toxicity of ox-
alic acid, 190. Potassium chlo-
rate in blood, 194. Cytisine in
urine, 206. Digitalis glucosides
and urine, 209. Toxic ergot resin,
209. Saponins on blood, 221.
Saponins solvents of cholesterin,
222. Toxicity of saponins, 222.
Extraction of solanine, 226.
Toxic action of solanine, 226.
Vegetable agglutinines, 228.
Crotin, 229. Toxic action of
piperine, 287.
Koenig, J., Piperine in pepper, 288.
Koenigs, W., Structure of quinine, 119.
de Konink, Detection of potassium,
193-
Koppeschaar, Estimation of carbolic
acid, 31.
Kossler, Estimation of cafbolic acid,
33-
Kraft, F., Ergotinine, 209.
Kratter, Examination of charred
blood, 311.
Krauss, L., Iodine on potassium
xanthogenate, 49.
Kraut, Preparation of potassium bis-
muthous iodide reagent, 255, 318.
Kreis, H., Cholesterine and phyto-
sterine with Melzer's reagent, 68.
Kiister, W., Formula of haematin, 310.
Kunkel, Normal arsenic, 175. Chro-
mium poisoning, 177. Tin poi-
soning, 181.
Langley, Picrotoxin test, 68.
Lautenschlaeger, Morphine test, 136.
Laves, E., Furfural test for veratrine,
96.
Leach, Estimation of methyl alcohol,
55-
Legal, Acetone test, 56.
Lehmann, V., Determination of silver
in organs, 179. Zinc in the dog,
1 80. Toxic effect of sulphur di-
oxide in air, 188.
Leins, H., Separation and estimation
of caffeine and theobromine, 272.
Le Nobel, Modification of Legal's ace-
tone test, 56.
Lieben, lodoform test for ethyl alcohol,
50. Same for acetone, 56.
Link, Limit of delicacy of Prussian blue
test for hydrocyanic acid, 22.
Lloyd, J. U., Morphine test, 134.
Lockemann, G., Destruction of or-
ganic matter, 234. Precautions
in Marsh test, 236. Delicacy of
Marsh test, 237.
INDEX OF NAMES
327
Loretz, Estimation of pilocarpine, 286.
Ludwig, Estimation of chloroform, 38.
Lustgarten, Naphthol test for chloro-
form, 3^. lodoform test, 42.
Lythgoe, Estimation of methyl alco-
hoi, 55-
Maassen, A., Biological arsenic test,
243-
Madsen, Solution of cholesterin in
saponin solutions, 222.
Magnin, Alkaloids extracted from
animal matter, 62.
Mai, C., Destruction of organic matter,
152. Electrolytic estimation of
arsenic, 237. Electrodes in ar-
senic determinations, 239. De-
struction of organic matter in
arsenic determinations, 239.
Mandelin, Test for strychnine, 99.
For hydrastine, 118. For pilo-
carpine, 219. Preparation of re-
agent, 321.
Mann, Lead in urine and faeces, 176.
Marquis, Morphine test, 134. Mor-
phine in the organism, 137.
Marsh, Arsenic test, 156.
Matthes, H., Estimation of alkaloids
as picrolonates, 253. Hydrastine,
282. Pilocarpine, 286. Nuxvom-
ica alkaloids, 296.
Mauch, R., Chloral hydrate in alkaloid
tests, 88. In brucine test, 102.
In codeine test, 112. In toxi-
cological analysis, 251.
Mecke, Codeine test, 113. Reagent
for opium alkaloids, 214. Prepa-
ration of reagent, 214, 322.
Meine, W., Berberine, 282.
Melzer, H., Carbolic acid test, 30.
Picrotoxin, 67. Nicotine, 92.
Mensching, J., Interference with phos-
phorus test, 6.
Merck, E., Assaying officinal extracts,
302.
v. Mering, J., Conjugated chloral hy-
drate in urine, 41. Excretion. of
veronal, 81. Isolation of same
from urine, 82.
Messinger, Estimation of carbolic acid,
33-
Meyer, G., Solanine in potatoes, 291.
Meyer, H., Chloroform iu the body, 35.
Action of chloral hydrate, 40.
Meyer, R., Composition of picrotorin,
67. Percentage of solanine in
potatoes, 225.
Miller, W. V., Structure of quinine,
1 20.
Millon, Carbolic acid test, 28. Sali-
cylic acid test, 78. Preparation of
reagent, 321.
Mitscherlich, Phosphorus test, 5.
Estimation of phosphorus, 15.
Mockel, Limit of delicacy of Prussian
blue test for hydrocyanic acid, 22.
Molle, B., Excretion of veronal, 81.
Isolation from urine, 82.
v. Morgenstern, F., Solanine in pota-
toes, 292.
Morner, Karl Th., Estimation of
minute amounts of arsenic, 247.
Moufang, Structure of brucine, 101.
Mulliken, Methyl alcohol test, 53.
Musculus, Conjugated chloral hydrate
in urine, 41.
Mylius, Fr., Preparation of pure iodeo-
sine, 322.
Nattermann, Phosphorus test, n.
Reduction of phosphorous acid to
phosphine, 14. Apparatus for es-
timating phosphorus, 15. Sensi-
tiveness of Mitscherlich test, 16.
Nessler, Chloral hydrate test, 39-
Preparation of reagent, 321.
Neubauer, Phosphorus test, 10.
Neumann, Detection of phosphorus in
cadavers, 16.
Nietzki, Composition of isopurpuric
acid, 71.
Noguchi, Solution of cholesterin in
saponin solutions, 222.
328
INDEX OF NAMES
Oliver, Lead in the organism, 177.
Osann, Maltol, 251.
Page, Solubility of carbon disulphide,
46.
Pagel, Normal arsenic, 175.
Pagenstecher, Hydrocyanic acid test,
21.
Palet, Pyramidone test, 126. Apo-
morphine test, 129.
Palm, R., Detection of ergot in flour,
211.
Panchaud, Estimation of cinchona al-
kaloids, 266.
Parmentier, Solubility of carbon disul-
phide, 46.
Partheil, A., Identity of cytisine and
ulexine, 205.
P61igot, Solubility of carbon disul-
phide, 46.
Pellagri, Codeine test, 112. Apo-
morphine, 128. Morphine, 134.
Penny, Estimation of carbolic acid, 33.
Penzoldt, Acetone test, 57.
Petri, Composition of isopurpuric acid,
7i.
Piccard, J., Ortho-xylene from can-
tharidin, 204.
Piccini, Isomethyl-pelletierine, 270.
Pictet, A., Nicotine synthesis, 91.
Pinner, Nicotine formula, 91. Nitro-
gen in pilocarpine and isopilo-
carpine, 218.
Pisani, Cocaine test, 109.
Pohl, Chloroform in blood and in the
body, 35.
Polacci, E., Procedure in thalleioquin
test, 121.
Poleck, Arsenic test, 163.
Polstorff, K., Interference with phos-
phorus test, 6.
Pouchet, Antimony in the body, 176.
Prescott, A. B., Estimation of alka-
loids, 257.
Proells, Cocaine in the cadaver, 107.
Pschorr, R., Apomorphine structure,
127. Morphol synthesis, 132.
Morphine structure, 133. The-
baine structure, 227. Thebaol
synthesis, 227.
R
Radulescu, D., Morphine test, 136.
Rammstedt, O., Estimation of alka-
loids as picrolonates, 253. Hy-
drastine, 282. Pilocarpine, 286.
Nux vomica alkaloids, 296.
Ramverda, Cytisine test, 206.
Ransam, Influence on saponins of
cholesterin in blood, 222.
Reichard, C., Cocaine test, 109.
Reynolds, Acetone test, 57.
Riechelmann, Estimation of caffeine
in coffee, 272.
Riegel, Estimation of antimony by
Gutzeit method, 240.
Roser, Narcotine structure, 114.
Pseudo-narceine from narcotine,
138.
Rossi, Conversion of nitrobenzene in
organism to aniline, 43.
Roussin, Nicotine test, 92.
Riigheimer, Piperine synthesis, 287.
Rupp, E., Iodine on xanthogenate, 49.
Russanow, A., Formula of compound
from phenol and benzaldehyde,
Sanger, C. R., Estimation of ar-
senic and antimony by Gutzeit
method, 240.
Schaefer, Normal arsenic, 175.
Schaer, E., Saponin test, 223. Animal
and vegetable catalysts and oxy-
gen carriers, 312. Blood stain
test, 313. Aloin blood test, 314.
Schaffer, Conjugated sulphuric acid
in urine in carbolic acid poisoning,
27.
Scherer, Phosphorus test, 3. Estima-
tion of same, 15.
Schiff , Reagent for aldehydes, 54.
Schindelmeiser, Nicotine test, 93.
INDEX OF NAMES
329
Schmidt, E., Crystalline veratrine, 94.
Apomorphine tests, 129. Detec-
tion of cantharidin, 205. Iso-
lation of schlererythrin, 210.
Detection of solanine and solarii-
dine, 226. Test for solanine and
solanidine, 227. Solubilities of
sulphates of cinchona alkaloids,
268. Estimation of berberine,
282. Estimation of alkaloids in
belladonna, etc., 300.
Schmiedeberg, O., Percentage of sola-
nine in potatoes, 225. Solanine
in potatoes, 291.
Schmitt, R., Preparation of salicylic
acid, 77.
Schneider, Sodium cacodylate not de-
composed by hydrochloric acid,
246.
Schonbein, Hydrocyanic acid test, 21.
Ozone test for blood, 312.
Scholtz, M., Detection of potassium
chlorate, 196.
Schiitze, Biological blood test, 315.
Schwarz, Resorcinol test for chloro-
form, 36.
Scudder, Methyl alcohol test, 53.
Selmi, Organic phosphorus compounds
in phosphorus test, 13. Pto-
maine resembling morphine, 220.
Silber, Structure of pseudo-pelletier-
ine, 270.
Simmonds, Methyl alcohol test, 54-
Skraup, Quinine structure, 120.
Socoloff, Caffeine in coffee, 275.
Sonnenschein, Destruction of organic
matter, 151.
Stas-Otto, Process for extracting alka-
loids, 63.
Stich, C., Estimation of phosphorus in
phosphorated oils, 232.
Strassmann, Ethyl alcohol eliminated
by lungs, 49.
Straub, W., Test for phosphorus in
phosphorated oils, 14. Estima-
tion of phosphorus in same, 231.
Accuracy of method, 232.
Strzyzowski, Sodium iodide in haemin
test, 308.
Tafel, Strychnine structure, 97. Bi-
chromate test for same explained,
99. Structure of brucine, loi.
Tanret, Ergotinine, 209.
Tassily, E., Caffeine in coffee, 272.
Teichmann, Haemin blood test, 309.
Thater, K., Santonin test, 200. San-
tonin in wormseed, 289.
Thiele, Compound of bromine and
phenol, 29.
Thorns, H., Separation of quinine from
mixtures, 122. Destruction of
organic matter, 150. Alkaloids
estimated by potassium bismu-
thous iodide, 255.
Thorpe, T. E., Isolation of methyl al-
cohol, 53. Arsenic in beer worts,
237-
Tollens, B., Formaldehyde in blood
test, 307.
Toth, J., Nicotine in tobacco, 280.
Trillich, H., Caffeine, 275.
Trotmann, Arsenic determined in beer
worts, 237.
Tiimmel, Arsenic test, 163.
U
Uhlenhuth, Biological blood test, 315.
Ulenberger, Lead in sheep, 17 7-
Ungar, Tin poisoning, 181.
Van Deen, Ozone blood test, 31.1.
Van der Moer, Cytisine test, 206.
Vandevelde, Toxicity estimated by
blood haemolysis, 258.
Vaubel, Solubility of antimony spot,
160.
Vitali, Carbon disulphide test, 47
Ethyl alcohol test, 51. Vera-
trine test, 96. Atropine test, 104.
Detection of caustic alkalies, 193
Blood stain test, 313.
330
INDEX OF NAMES
Volhard, Estimation of hydrochloric
acid, 184.
Von Pohl, Methyl alcohol in the or-
ganism, 52.
Vortmann, Hydrocyanic acid test, 23.
Estimation of carbolic acid, 33.
W
Wangerin, Narcotine test, 115. Apo-
morphine test, 128. Lloyd's mor-
phine test, 135. Narceine test,
140.
Warren, L. E., Papaverine test, 217.
Warren, W. H., 'Alkaloids purified by
picrolonic acid, 89. Normal ar-
senic, 176.
Wassermann, Biological blood test, 24.
Weehuizen, Hydrocyanic acid test, 24.
Weiss, R. S., Alkaloids purified by pic-
rolonic acid, 89.
Weppen, Veratrine test, 95.
Werk, R., Cause of solanine in pota
toeS, 22.S.
White, Tin poisoning, 181.
Wieser, Normal arsenic, 176.
Willstatter, Cocaine structure, 106.
Windhaus, Digitonin, 207. Saponin
cholesterides, 222.
Wittmann, Solanidine from solanine,
225.
Witz, Determination of mercury in the
body, 179.
Wolff, C., Caffeine in coffee, 276.
Zappi, Extraction of alkaloids from
animal matter, 62.
Zeine, Formula of picrolonic acid, 253.
Zeisel, Colchicin test, 69. Deter-
mination of methoxyl groups in
alkaloids, 101.
Ziemke, Normal arsenic, 1 76.
INDEX OF SUBJECTS
Abortifacients, Ergot, 209. Nitro-
benzene, 42.
Abrin, 229.
Absorption-spectra, blood, 306, 310.
Carmine, 311. Codeine, 112.
Ergot, 211. Fuchsine, 312.
Nitrobenzene, 42. Physos-
tigmine, no.
Acetanilide, 66, 73, 141.
Acetic ether (see Ethyl acetate).
Acetone, 55.
Acetonuria, 55.
Acid acetic, Aconitine, 262.
aconitic, 261.
amino-acetic, 58.
a-amino-isobutyl-acetic, 17.
angelic, 94.
arsenic, 150.
atropic, 103.
benzoic, 58. Aconitine, 262.
Cocaine, 106.
brucic, 101.
cacodylic, 246. Electrolysis, 234.
cantharic, 204.
cantharidic, 203, 263.
carbolic, 26. Aniline, 34. Esti-
mation, 31. Urine, 34.
chloric, Organic matter, 151. Re-
duction, 196.
chloroplatinic, 318. Potassium,
193-
chromic, Alcohol, 51. Aniline,
45. Cocaine, 108.
cincholoiponic, 119.
cinchonic, 119.
ethyl-sulphonic, 202.
ethyl-sulphuric, Solanine and
solanidine, 227.
Acid formic, Chloral hydrate, 39.
Phosphorus, 4.
glycuronic, 41, 44. Aceto-p-
amino-phenol, 74. Mor-
phine, 137. Organism, 123.
guaiaconic, 314.
hippuric, 58.
hydrastic, 117.
hydrochloric, 183. Colchicin, 69.
Digitalin, 209. Electrolytic,
241. Organic matter, 151.
Vera trine, 95.
hydrocyanic, 19. Blood, 312.
Estimation, 25. Oil of bitter
almonds, 58. Potassium
ferrocyanide, 25. Prelimi-
nary test, 21.
hydro-p-cumaric, 17, 28.
hypophosphorous, 8.
iodic, 321. Morphine, 134.
isopurpuric, 71.
lactic, Hydrocyanic acid, 20.
loiponic, 119.
mandelic, 105.
meconic, 213.
meconinic, 214.
n-methyl-granatic, 270.
Acid nicotinic, 91.
nitric, 184. Antipyrine, 83.
Apomorphine, 128. Co-
caine, 107. Codeine, in.
Colchicin, 69. Cytisine, 207.
Morphine, 133. Narceine,
139. Papaverine, 217.
Phenacerine, 76- Physostig-
mine,no. Pyramidone, 125.
Thebaine, 228. Veratrine,
95-
nitrous, Antipyrine, 83.
nor-meta-hemipinic, 117-
331
332
INDEX
Acid opianic, Hydrastine, 117. Nar-
cotine, 114.
oxalic, 189.
p-amino-phenyl-sulphuric, 44.
p-hydroxy-benzoic, 28.
p-oxy-phenyl-acetic, 17, 28.
p - oxy - phenyl -a- amino - pro-
pionic, 17.
p-oxy-phenyl-propionic, 17, 28.
phenyl-glycuronic, 26.
phospho-molybdic, 319.
phosphoric, Reduction, 8.
phosphorous, 8, 14. Mercury,
166.
phospho-tungstic, 319.
picramic, Organism, 70. Picric
acid, 24, 72.
picric, 66, 70, 141. Hydrocyanic
acid, 24. Reagent, 320.
picrolonic, 320. Alkaloids esti-
mated, 253; purified, 89.
Hydrastine, 282. Nux vo-
mica, 296. Pilocarpine, 286.
piperic, 287.
quinic, 119, 265.
quino-tannic, 265.
rubazonic, 124.
salicylic, 66, 77, 142. Foods and
beverages, 250. Phenols, 28,
78. _
salicyluric, 79.
santonic, 198, 290.
sarcolactic, 17.
sclerotic, 209
selenious, 113.
sphacelic/209.
strychnic, 97.
suberic, 270.
sulphanilic, 136.
sulphocyanic, 19.
sulphuric, 186. Apomorphine,
128. Cocaine, 107. Code-
ine, in. Colchicin, 69.
Conjugations, 74. Digitalin,
208. Hydrastine, 118. Me-
conine, 214. Narceine, 139.
Narcotine, 115. Papaverine,
217. Physostigmine, no.
Preformed and conjugate,
26. Santonin, 200. Saponins,
223. Solanine, 227. The-
baine, 228. Veratrine, 95.
sulphurous, 1 88.
tannic, 319. Antipyrine, 83.
Blood, 305. Caffeine, 85.
Colchicin, 69. Narceine,
140.
thioacetic, 249.
tiglic, 94.
trichlor-ethyl-glycuronic, 41.
trimethyl-colchicinic, 69.
tropic, 103, 1 06.
uric, 84.
urochloralic (see Trichlor-ethyl-
glycuronic).
vanadic-sulphuric, 226.
veratric, 216.
Acid xanthoproteic, 185.
Acids, mineral, 182.
Aconine, 262.
Aconitine, 261.
Agglutinines, 228.
Air, Carbon disulphide, 48. Sul-
phur dioxide, 188.
Albuminates, metallic, 151, 172.
Alcohol, ethyl, 49. Differentiation,
57. Purification, 61. Tox-
icity, 259.
methyl, 52. Cocaine, 106. Col-
chicin, 68. Estimation, 55.
Toxicity, 259.
trichlor-ethyl, 41.
Alkalies, 192.
Alkaloidal reagents (see Reagents,
alkaloidal).
Alkaloids, Aconite, 261. Alkaloidal
reagents, 87. Belladonna,
301. Cinchona, 264. Esti-
mation, 253, 255, 257, 296.
Extraction, 86. Extracts,
266. Hyoscyamus, 301.
Ipecac, 277. Nux vomica,
293-295. Physiological test,
88. Pomegranate, 270. Puri-
fication, 88. Selenious-sul-
phuric acid, 215. Viscera, 62.
INDEX
333
Aloes, Quinine test, 122.
Aloin, Blood test, 314.
Aluminium acetate, 289.
Ammonia, 192. Fapaverine, 217.
Physostigmine, no.
Ammoniacal copper, Picric acid, 72.
Ammoniacal silver, Morphine, 135.
Picrotoxin, 67.
Ammonium magnesium phosphate, 7.
molybdate, 7.
persulphate, 152.
sulphide, Blood, 305. Sulphides,
i54-
Anhydro-ecgonine, 106.
Aniline, 44, 93, 143. Carbolic acid,
34-
Antimony, 155. Detection, 163, 165.
Distribution, 175. Estima-
tion, 240. Differentiation of
spot, 1 60.
Antipyrine, 66, 82, 87, 123, 142, 145,
147. Extract, 87. Thalleio-
quin, 121. Urine, 83, 123.
Antipyryl-urea, 125.
Apomorphine, 127, 146. Selenious-
sulphuric acid, 215.
Argyria, 179.
Arrhenal, 246. Electrolysis, 234.
Urine, 247.
Arsenic, 155. Beer, 237. Betten-
dorff, 162. Biological, 242.
Bougault's reagent, 247.
Bulb-tube, 162. Differentia-
tion, 1 60. Distribution, 173.
Electrolysis, 233, 237. Eli-
mination, 173. Fresenius-v.
Babo, 161. Gutzeit, 163,
240. Isolation, 233. Locke-
mann, 234. Marsh-Berze-
lius, 156. Morner, 247.
Normal, 174. Organic com-
pounds, 245. Spot, 157.
Urine, 247.
Arsenic trichloride, 150. Arsenic iso-
lated, 233.
Arseno-tungstic reagent, 125.
Arseno-tungsto-molybdic reagent, 125.
Arsine, 156.
Asparagine, 292.
Atoxyl, 246. Electrolysis, 234.
Urine, 247.
Atropa belladonna, Alkaloids, 30x3.
Atropine, 87, 102, 144. Estimation,
300, 302. Extract, 87.
General reagents, 105.
Physiological test, 104.
Putrefaction, 103.
Barbital, 79
Barium, 170.
Baumert's nitric acid test, 185.
Beer, Arsenic, 237. Picrotoxin, 68.
Saponins, 223.
Benzaldehyde, 57. Phenol, 30.
o-nitro, 57.
p-dimethyl-amino, 104.
Benzaurine, 31.
Benzoyl cocaine test, 108.
Benzoyl-ecgonine, 106.
Berberine, 116. Estimation, 282.
Berthelot's alcohol test, 50.
Bettendorff's reagent, 322. Test, 162.
Biological test, Arsenic, 242. Blood,
3*4-
Bile, Elimination of lead, 177.
Bismuth, 165. Elimination, 180.
Morphine, 135. Physiolog-
ical action, 172. Toxicity,
1 80.
Bitter almond water, 57.
Blondlot-Dusart test, 8.
Blood, Acetone, 55. Aniline, 44.
Carbon disulphide, 46. Car-
bon monoxide, 304. Chloral
hydrate, 40. Chloroform,
35. Copper, 178. Defibrin-
ated, 229. Heavy metals,
173. Hydrocyanic acid, 20.
Lead, 177. Nitrobenzene,
42. Oxalates, 190. Phos-
phorus, 17. Picric acid, 70.
Potassium chlorate, 194.
Saponins, 221. Stains, 307,
313. Sulphurous acid, 188.
334
INDEX
Tests, 306, 310, 312, 314.
Toxalbumins, 228.
Boiling test for blood, 304.
Bread, Ergot, 211.
Bromine test, Aniline, 45. Digitonin,
208. Phenol, 28. Pyrami-
done, 125. Salicylic acid, 78.
Brucine, 86, 100, 143. Estimation,
296. Extract, 86. General
reagents, 101. Nitric acid
test, 186. Picrolonate, 296.
Tests, 102.
Bulb-tube test, Arsenic, 162.
Cacao, Caffeine and theobromine, 298.
Cadmium, 167.
Caffeine, 66, 83, 87, 122, 142, 145, 147.
Estimation, 271, 274, 298.
Extract, 87. Extraction, 275.
Fate, 84. Tests, 85. Thal-
leioquin test, 121.
Calabar bean, no.
Canadine, 116.
Cantharene, 204.
Cantharic anhydride, acetyl-hydrato,
204.
Cantharidin, 142, 203. Estimation,
263.
Carbolic urine, 26.
Carbon disulphide, 46. Air, 48.
Tests, 47.
Carbon monoxide blood, 304.
Carbon tetrachloride, Caffeine, 274.
Purification, 275. Separa-
tion of theobromine, 299.
Carboxyhaemoglobin, 304.
Carmine, Absorption-bands, 311.
Castor bean, 229.
Cephaeline, 277. Test, 278.
Cevadine, 93.
Cevine, 94.
Chavicine, 287.
Chloral hydrate, 38. Blood, 313.
Decomposition, 39. Estima-
tion, 41. Fate, 40. Physio-
logical action, 40. Powders,
40. Saponin test, 223. Sol-
vent, 251. Tests, 39.
Toxicological analysis, 40.
Chlorine-ammonia test, Narceine, 139.
Chlorine test, Hydrochloric acid, 183.
Potassium chlorate, 195.
Chlorine water, Preparation, 85.
Thebaine, 228.
Chloroform, 35. Estimation, 38.
Tests, 36.
Chocolate, Caffeine, 298.
Cholesterine, Haemolysis, 222, 224.
Melzer's reagent, 68. Sapo-
nins, 221.
Choline, Ergot, 211.
Chrome yellow test, 170.
Chromium, 169, 177.
Cicutoxine, 66.
Cinchona, Alkaloids, 264. Quinine,
268.
Cinchonidine, 265.
Cinchonine, 119.
Claviceps purpurea, 209.
Cocaine, 105, 144. Organism, 107.
Physiological test, 109. Re-
lation to atropine, 106.
Tests, 107.
Cocculus indicus, 66.
Codeine, 87, in, 144. Estimation,
254. Extract, 87. Selen-
ious acid, 215. Tests, in.
Coffee, Caffeine, 271.
Cola nuts; Caffeine, 273, 276.
Colchicein, 68.
Colchicin, 65, 68, 141. Estimation,
269. Hydrolysis, 68. Puri-
fication, 70. Structure, 69.
Tests, 69.
Colchicum autumnale, 68. Colchicin,
269.
Congo paper, 182.
Conhydrine, 89.
7-Coniceine, 89.
Coniine, 89, 142. Diazonium ' test,
137. Tests, 90.
Conium maculatum, 89.
Conjugated aceto-p-amino-phenol, 74.
p-Amino-phenol, 44. Anti-
INDEX
335
pyrine, 123. Benzoic acid,
58. Carbolic acid, 26.
Chloral hydrate, 41. Mor-
phine, 137. Salicylic acid,
79-
Constitution, Acetanilide, 73. Anti-
pyrine, 82. Apomorphine,
127. Atropine, 103. Caf-
feine, 83. Cantharidin, 203.
Cocaine, 105. Codeine, in.
Coniine, 89. Homatropine.
105. Hydrastine, 117,
Morphine, 131. Narceine.
138. Narcotine, 113. Nico,
tine, 91. Papaverine, 215-
Phenacetine, 75. Picric acid,
70. Piperine, 288. Pseudo-
pelletierine, 2 70. Quinine,
119. Salicylic acid, 77.
Santonin, 198. Thebaine,
227. Veratrine, 94. Vero-
nal, 79.
Copper, 155, 165, 167. Elimination,
178. Fusion, 155. Mercury
test, 1 66. Nitric acid test,
1 86. Physiological action,
172, 178. Tests, 155, 167.
Toxicity, 172.
oxide, 156.
sulphate, Blood test, 305. Phos-
phorus, 14.
sulphide, Solubility, 155.
Corn smut, 211.
Cornutine, 209.
Corrosion, 173.
Cotarnine, 114, 118.
Couerbe's test, Narcotine, 115.
Creatinine, 87.
Cresols, 28.
Crotin, 229.
Croton Tiglium, 229.
Crystallization test, Coniine, 90. Nic-
otine, 92. Oxalic acid, 191.
Cyanogen, 24.
Cyano-haemoglobin, 20.
Cystine, 17.
Cytisine, 205. Tests, 206.
Cytisus Laburnum, 205.
Datura strammonium, Alkaloids, 300.
Deniges test, Cocaine, 109.
Destruction of organic matter, 148,
150-152.
Dextrose, Digitalin, 208. Digitonin,
207. Hydrocyanic acid, 20.
Phosphorus, 17.
Dialysis, Potassium chlorate, 194.
Diazonium test, Morphine, 136.
Dichromate test, Strychnine, 98.
Digitalis glucosides, 207.
Dimethoxy-isoquinoline, 216.
p-Dimethyl-amino-benzaldehyde, 104.
Dionine, 136.
Diphenylamine test, Nitric acid, 186.
Reagent, 186, 235.
Distillation test, Ammonia, 192. Hy-
drochloric acid, 183. Nitric
acid, 184.
Distribution, Antimony, 175. Ar-
senic, 173. Carbolic acid,
27. Chloroform, 35, 38.
Ethyl alcohol, 49. Heavy
metals, 173. Hydrocyanic
acid, 20. Mercury, 178.
Oxalic acid, 190. Tin, 181.
Zinc, 1 80.
Diuresis, 194.
Dyeing test, Picric acid, 72.
E
Ecgonine,' 106.
Electrolysis, Arsenic compounds, 233,
237-
Elimination, Antimony, 175. Arsenic,
173. Bismuth, 180. Chro-
mium, 178. Cocaine, 107.
Copper, 178. Heavy metals,
173. Hydrocyanic acid, 19-
Lead, 176. Mercury, 178.
Picric acid, ?*> Potassium
chlorate, 194- Strychnine,
97. Tin, 181. Veronal, 81.
Emetine, 277. Diazonium test, 137.
Epichlorohydrin, 92.
336
INDEX
Erdmann's reagent, 320. Cocaine,
107. Colchicin, 69. Nar-
ceine, 139. Narcotine, 115.
Papaverine, 217. Strych-
nine, 98. Thebaine, 228.
Veratrine, 95.
Ergot, 209.
Ergotine, 209.
Ergotinine, 209. Estimation, 211.
Test, 212.
Ergotoxine, 209.
Erythrosine (see lodeosine).
Eserine (see Physostigmine).
Ether extracts, evaporation, 64.
Ethyl acetate, Alcohol, 51. Caffeine,
275-
Exhaustive methylation, 117. n-
Methyl-granatic acid, 270.
Extract belladonna, Alkaloids, 301,
302.
cinchona, Alkaloids, 266, 302.
Quinine, 269.
hydrastis, Hydrastine, 281, 282.
Berberine, 282.
hyoscyamus, 301.
nux vomica, Alkaloids, 295, 296,
303-
opium, Morphine, 285.
F s
Faeces, lead in, 176.
Fat, Phosphorus, 17.
Fate, Acetanilide, 74. Benzaldehyde,
58. Caffeine, 84-. Chloral
hydrate, 40. Cocaine, 107.
Digitalis glucosides, 209.
Ethyl alcohol, 49. Heavy
metals, 173.
Fehling's solution, 320. Chloroform,
37. Githagin, 224. Maltol,
251. Picrotoxin, 67. Sapo-
nins, 224.
Ferments, Hydrocyanic acid, 20.
Ferric chloride, Antipyrine, 83. Apo-
morphine, 129. Benzoic
acid, 58. Carbolic acid, 29.
Codeine, 112. Cytisine, 206.
Ergotinine, 212. Maltol,
251. Meconic acid, 213.
Morphine, 134. Pyrami-
done, 125. Salicylic acid,
77, 250, 251. Santonin, 200.
Ferric hydroxide, Arsenic, 235.
Ferrous sulphate test, Nitric acid, 186.
Fleury's test, Morphine, 135. Nitric
acid, 185.
Flour, Ergot, 211. Githagin, 223.
Fluorescence test, Hydrastine, 118.
Quinine, 120.
Formaldehyde, Blood, 307. Phos-
phorus, 4.
Formaldehyde-sulphuric acid, 321.
Codeine, 112. Morphine,
134. Pilocarpine, 219.
Formamide test, Cocaine, 109.
Fowler's solution, 247.
Fresenius-v. Babo, Arsenic, 161. Or-
ganic matter, 148.
Froehde's reagent, 320. Apomor-
phine, 128. Cephaeline, 278.
Cocaine, 107. Codeine, 112.
Emetine, 279. Hydrastine,
118. Morphine, 134. Nar-
ceine, 139. Narcotine, 115.
Papaverine, 217. Saponins,
222. Solanine, 227. Strych-
nine, 98. Thebaine, 228.
Veratrine, 95.
Fuchsine, Absorption-band, 312.
Furfural, Codeine, 112. Santonin,
200.
Fusion of heavy metals, 155.
Galactose, Digitonin, 207. Solanine,
225.
General alkaloidal reagents (see Re-
agents, general).
German Pharmacopoeia (see Pharma-
copeia, German).
Githagin in flour, 223.
Glucose (see Dextrose).
Glycocoll (see Acid, amino-acetic).
Glycosuria, 17.
INDEX
337
Glycurone, 44.
Glyoxaline, 218.
Gold chloride, 317.
Golden chain, 205.
Grandeau's test, Veratrine, 96.
Guaiac-copper paper, 2 1 .
Guaiac resin, Blood, 312.
Guarana, Caffeine, 276.
Guglialmelli's test, Pyramidone, 125.
Giinzburg's reagent, 321. Mineral
acid, 182.
Gutzeit's test, 163, 240.
H
Haematin, 310.
Haematin, reduced (see Haemochro-
mogen).
Haematoporphyrin, Spectrum, 311.
Urine, 202.
Haematoxylin, 266, 303.
Haemin, 310.
Haemin crystals, 308.
Haemochromogen, 310.
Haemoglobin with metals, 173.
Haemoglobinuria, 226.
Haemolysis, 224. Cholesterin, 222.
Saponins, 221.
Herapathite test, Quinine, 120.
Heroine, 136.
Hirschsohn's test, Quinine, 122.
Homatropine, 105.
Hunef eld's solution, 321.
Husemann's test, Morphine, 133.
Hydrastal, 117.
Hydrastine, 116, 144. Estimation,
281.
Hydrastinine, 117.
Hydrastis canadensis, 116.
Hydrocotarnine, 114.
Hydro-ergotinine, 209.
Hydrogen sulphide, arsenic-free, 152.
Phosphorus, 3.
Hydrolysis, Aconitine, 262. Atropine,
103. Cocaine, 106. Colchi-
cin,68. Digitalin, 208. Digi-
tonin, 207. Digitoxin, 208.
Narcotine, 114. Piperine,
22
287. Saponins, 221. Sola-
nine, 225.
Hydroquinol, 26.
Hyoscyamine, 103.
Hyoscyamus niger, Alkaloids, 300.
Hyper-isotonic solutions, 224.
Hyp-isotonic solutions, 224.
Hypochlorite test, Acetanilide, 74.
Aniline, 45. Carbolic acid,
29.
1
Ignatius beans, 96.
Indigo test, Chloric acid, 195.
Indigo tine test, Acetone, 57.
Indophenol test, Acetanilide, 73.
Phenacetine, 76.
lodeosine, 322.
Iodine test, Narceine, 139. Pyrami-
done, 125.
lodoform, 41.
lodoform test, Acetone, 56.
lodo-potassium iodide, 318. Alka-
loids, 257.
Ipecac, Alkaloids, 277, 279.
Isomethyl-pelletierine, 270.
Iso-pelletierine, 270.
Iso-pilocarpine, 217.
Isopurpuric acid test, Picric acid, 71.
Iso-quinoline, 216.
Isotonic solutions, 224.
J
Jaborandum, Alkaloids, 217. Pilo-
carpine, 286.
Jaborine, 217.
Jequirity seeds, 229.
Kiliani's test, Digitoxin, 208.
Kjeldahl, Caffeine estimation, 274.
de Konink's reagent, 193.
Langley's test, Picrotoxin, 68.
Lead, 167. Blood, 173- Elimination,
338
INDEX
176. Sheep, 177. Physio-
logical action, 172.
Lead acetate test, Blood, 304. Car-
bon disulphide, 47.
Lead paper, Phosphorus, 3.
Lecithine-saponins, 221.
Leucine, Phosphorus, 17.
Leucocytosis, 46.
Lichen's test, Alcohol, 50. Acetone,
56.
Liver, Copper, 178.
Lloyd's test, Morphine, 134.
Lustgarten's test, lodoform, 42.
Lytta vesicatoria, 203.
Magnesia mixture, 7, 321.
Mai's method, Organic matter, 152.
Maltol, 251.
Mandelin's reagent, 321. Hydrastine,
1 1 8. Pilocarpine, 219.
Strychnine, 99.
Marquis' reagent, 134.
Marsh apparatus, 134.
Marsh-Berzelius test, 156, 236.
Mat6 (see Paraguay tea).
Material to be examined, Arsenic, 173.
Bismuth, 180. Carbolic
acid, 26, 27. Chloroform,
38. Copper, 178. Digitalis
glucosides, 209. Ethyl alco-
1 hoi, 49. Hydrocyanic acid,
acid, 21. Mercury, 178.
Potassium chlorate, 194.
Veronal, 81. Zinc, 180.
Mauch's solvent, 251. Brucine, 102.
Meadow saffron, 68.
Meat, Potassium chlorate, 197. Sali-
cylic acid, 250. Sulphur
dioxide, 189.
Mecke's reagent, 322. Codeine, 113.
Meconine, 213.
Melting-point test, Salicylic acid, 78.
Sulphonal, 201.
Melzer's reagent, 67. Carbolic acid,
30. Picrotoxin, 67.
Melzer's test. Nicotine, 92.
Menispermum cocculus, 66.
Mercuric chloride, 318.
cyanide, 25, 65.
iodide, Mercury, 166.
Mercury, 165. Distribution, 178.
Physiological action, 172.
Tests, 166. Tin, 164.
Toxicity, 172. Urine, 178.
Meroquinene, 120.
Metallic poisons (see Poisons, metallic).
Metals, heavy, 172.
Methaemoglobin, 308. Absorption-
band, 310.
n-Methyl-coniine, 89.
n-Methyl-granatoline, 270.
n-Methyl-granatonine (see Pseudo-
pelletierine).
Methyl orange, 182.
Methyl-pelletierine, 270.
Methyl violet, 182.
Milk, Salicylic acid, 251. Toxalbu-
mins, 228.
Millon's reagent, 321. Carbolic acid,
28. Maltol, 251. Salicylic
acid, 78.
Mitscherlich's phosphorus test, 5, 15.
Morphenol, 132.
Morphine, 131, 146. Estimation, 254.
General reagents, 137.
Opium, 283. Organism, 137.
Preliminary test, 130. Pu-
trefaction, 137. Selenious-
sulphuric acid, 215.
Morphol, 132.
Murexide reaction, 85.
N
Naphthol test, Chloroform, 36.
Narceine, 138, 147. Selenious-sul-
phuric acid, 215.
Narcotine, 113, 145. Selenious-sul-
phuric acid, 215.
Nessler's reagent, 321. Ammonia,
192. Chloral hydrate, 39.
Nicotine, 90, 143. Diazonium test,
137. Tobacco, 279.
Nitrite test, Carbolic acid, 30.
INDEX
339
Nitrobenzene, 42. Differentiation
58.
Nitro-phenacetine, 76.
Nitroprusside test, Acetone, 56. Hy-
drocyanic acid, 22.
Non- volatile poisons (see Poisons, non-
volatile).
Normal arsenic, 1 74.
Nux vomica, 96. Alkaloids, 293-296.
Odor test, Atropine, 104.
Oil of bitter almonds, Hydrocyanic
acid, 58.
Oils, phosphorated, Phosphorus, 14,
231-
Opium, 212. Morphine, 283. Mor-
phine in tincture, 285. Mor-
phine in wine, 285.
Organic arsenic compounds, 245.
Electrolysis, 234.
Organic matter, Destruction, 148, 151,
152, 234.
Oxalates in plants, 190.
Oxidation test, Methyl alcohol, 53-55.
Caffeine, 85. Codeine, in.
Phenacetine, 76. Picro-
toxin, 67.
Oxy-dimorphine, 133.
Oxy-haemoglobin, 304. Reduction,
37-
Oxy-santonins, 199.
Palet's test, Apomorphine, 129. Py-
ramidone, 126.
Palladous chloride test, Blood, 305.
Papaveraldine, 216.
Papaverine, 145, 215.
Paracholesterine, 68.
Paraguay tea, Caffeine, 276.
Paraxanthine, 84, 300.
Pellagri's test, Apomorphine, 128.
Codeine, 112. Morphine,
i34-
Pelletierine, 270.
Penzoldt's test, Acetone, 57.
Pepper, 287. Pipeline, 288.
Peptones, Phosphorus, 17. Animal
matter, 87.
Peptonuria, 17.
Peronine, 136.
Pharmacopoeia, German, Aconitine,
262. Alkaloids, 260. Bella-
donna, 301. Cantharidin,
263. Cinchona, 264. Hy-
drastine, 281. Hyoscyamus,
301. Ipecac, 279. Nux vom-
ica, 294, 295. Opium, 283.
Pomegranate, 271.
Phenacetine, 66, 75, 141.
Phenanthrene, 132.
Phenol (see Acid, carbolic).
Phenol, aceto-p-amino, 74.
Phenolphthalein, 24.
Phenolphthalin test, Hydrocyanic
acid, 24.
Phenylisocyanide test, Acetanilide, 73.
Aniline, 45. Chloroform, 36.
Phosphine, 8.
Phosphorous acid (see Acid, phos-
phorous).
Phosphorus, yellow, 5. Antidote, 14.
Blondlot-Dusart, 8. Frese-
nius, Neubauer, 10. Hilger-
Nattermann, n. Magnesium
test, 7. Metabolism, 16. Mit-
scherlich test, 5. Molybdate
test, 7. Oils, 14, 231. Or-
ganic compounds, 13.
Scherer's test, 3. Spectrum,
12. Urine, 17.
Physiological action, Acetanilide, 73.
Acetone, 55. Alkalies, 192.
Berberine, 282. Bismuth,
172. Cantharidin, 205.
Carbolic acid, 26.
Chloral hydrate, 4- Chlo-
roform, 35. Chromium, 177.
Copper, 172, 178- Ergot,
210. Heavy metals, 172-
Homatropine, 105. Hydro-
cyanic acid, 19. Lead, 172.
Mercury, 172. Morphine,
340
INDEX
137. Nicotine, 91. Nitric
acid, 184. Nitrobenzene, 42.
Oxalic acid, 190. Phos-
phorus, 16. Picric acid, 70.
Pyramidone, 124. Santonin,
199. Saponins, 221. Silver,
172, 179. Strychnine, 97.
Sulphuric acid, 186. Sul-
phurous acid, 188. Tin, 181.
Uranium, 172, 180. Vero-
nal, 80.
Physiological salt solution, 224.
Physiological test, Alkaloids, 88.
Atropine, 104. Cantharidin,
205. Cocaine, 109. Nico-
tine, 93. Physostigmine,
no. Strychnine, 99. Vera-
trine, 94.
Physostigma venenosum, no.
Physostigmine, no, 145. Diazonium
test, 137.
Phytosterine, 68, 281.
Picraconitine, 261.
Picro-sclerotine, 209.
Picrotin, 66.
Picrotoxin, 65, 66, 140.
Picrotoxinin, 66.
Pilocarpidine, 217.
'Pilocarpine, 217. Estimation, 286.
Pilocarpus pennatifolius (see Jaboran-
dum).
Piperidine, 287. Diazonium test, 137.
Piperine, 287. Estimation, 288.
Pisani's test, Cocaine, 109.
Platinum chloride, 318.
Poisons, metallic, 148.
non- volatile, 61.
volatile, 3. Distillation, 18.
Pomegranate bark, Alkaloids, 270.
Potassium arsenate, in.
bismuthous iodide, 318. Alka-
loids, 255.
cadmium iodide, 318.
chlorate, 194. Meat, 197. Or-
ganic matter destroyed, 148.
Putrefaction, 197. Urine, 197.
ferrocyanide, 25. Blood, 305.
Copper, 167.
Potassium hydroxide, 192. Santonin,
200.
mercuric iodide, 318. Alkaloids,
258.
permanganate, Cocaine, 108.
pyro-antimonate, Sodium, 193.
zinc iodide, 319.
Potatoes, Asparagine, 292. Sola-
nine, 291.
Precipitation test, Cocaine, 107.
Copper, 167.
Proteins, Phosphorus, 17. Chloro-
form, 35. Heavy metals,
172.
Prussian blue test, Hydrocyanic acid,
22. Morphine, 135. Tin,
164.
Pseudo-conhydrine, 89.
Pseudo-morphine (see Oxy-dimor-
phine).
Pseudo-narceine, 138.
Pseudo-pelletierine, 270.
Psychotrine, 277.
Ptomaines, 219.
Putrefaction, Carbolic acid, 27. Atro-
pine, 103. Cantharidin, 205.
Morphine, 137. Potassium
chlorate, 197. Phosphorus,
16.
Pyramidone, 87, 124, 145. Extract,
87.
Pyridine, Chloroform, 37. Coffee,
275-
Pyrocatechol, 26.
Pyrone, 213.
Quinidine, 264.
Quinine, 87, 119, 145. Estimation,
258, 268. Extract, 87.
Quinoline, 216.
Radulescu's test, Morphine, 136.
Ramverda's test, Cytisine, 206.
Reagents, general alkaloidal, 317.
INDEX
341
Resorcinol test, Chloroform, 36. Nar-
ceine, 140.
Reynold's test, Acetone, 57.
Rhamnose, 225.
Ricin, 229.
Roussin's test, Nicotine, 92.
Rubreserine test, Physostigmine, no.
Santonin, 198. Estimation, 289.
Sapogenins, 221.
Saponins, 220. Beer, 223. Choles-
te rides, 222.
Schaer's test, Blood, 313, 314.
Scherer's test, Phosphorus, 3.
Schindelmeiser's test, Nicotine, 93.
Schlererythrin, 209. Test, 211.
Schmidt's test, Apomorphine, 129.
Schonbein-Pagenstecher test, Hydro-
cyanic acid, 21.
Schonbein-Van Been test, Blood, 312.
Scopolamine, 104.
Secale cornutum, 209.
Selenic-sulphuric acid test, Solanine,
226.
Selenious-sulphuric acid, 214. Narco-
tine, 116. Papaverine, 217.
Selenium, Moulds, 243.
Silver, 171. Estimation, 179. Or-
ganism, 179. Physiological
action, 172.
Silver nitrate test, Hydrocyanic acid,
23. Potassium chlorate, 195.
Silver phosphide, 3, 9.
Sodium arsenate, 156.
hydroxide, 192. Blood, 304.
iodide, Haemin crystals, 308.
nitroprusside, 56.
perchlorate, Cocaine, 109.
pyro-antimonate, 156.
stannate, 156.
thiosulphate, Chloral hydrate, 39.
Solanidine, 225. Test, 226.
Solanine, 225. Estimation, 291.
Test, 226.
Solanum tuberosum, 225.
Solubility test, Coniine, 90.
Sonnenschein-Jesserich, Destruction of
organic matter, 151.
Spanish flies, 263.
Sparteine, 137.
Spectroscopic test, Barium, 171.
Blood, 306, 310. Hydrogen,
12. Phosphorus, 12.
Spotted hemlock, 89.
Stannic oxide, 156.
Stannous chloride, 102, 322. Mer-
cury, 1 66.
Starch test, Sulphur dioxide, 189.
Stas-Otto process, 63.
Stibine, 160.
Straub's test, Phosphorus, 14.
Strychnine, 86, 96, 143. Estimation,
258, 298. Extract, 86. Pi-
crolonate, 296.
Stypticine, 254.
Sugar test, Sulphuric acid, 187.
Sulphocyanate test, Carbon disul-
phide, 47. Hydrocyanic
acid, 22. Mineral acid, 183.
Sulphonal, 200. Extraction, 198.
Sulphur dioxide test, Sulphuric acid,
187. Meat, 189.
Synopsis of Group I, 59.
Group II, 140.
Group III, 171.
Synthesis, Acetanilide, 73. Antipy-
rine, 82. Nicotine, 91.
Phenacetine, 75. Piperine,
287. Pyramidone, 1 24.
Salicylic acid, 77. Sulpho-
nal, 201. Veronal, 80.
Tea, Caffeine, 272, 274, 276. Theo-
phylline, 300.
Teichmann's crystals (see Haemin
crystals).
Tellurium, Moulds, 243.
Tetra-chloro-methane (see Carbon te-
trachloride).
Thalleioquin test, Quinine, 120.
Thebaine, 146, 227. Selenious-sul-
phuric acid, 215.
342
INDEX
Thebaol, 227.
Theine (see Caffeine).
Theobromine, 84, 299, 300. Estima-
tion, 277, 298.
Theophylline, 84, 300.
Thyroid gland, Arsenic, 174.
Tin, 155. Distribution, 181. Tests, 164.
Tincture of nux vomica, Alkaloids,
295, 297.
opium, Morphine, 285.
Tobacco, Nicotine, 278, 280.
Toxalbumins, 228.
Toxicity, Acetone, 55. Aniline, 44.
Bismuth, 180. Carbolic
acid, 26. Carbon disulphide,
46. Chloral hydrate, 40.
Chromium, 177. Copper,
172. Cytisine, 206. Esti-
mation by haemolysis, 258.
Ethyl alcohol, 259. Homa-
tropine, 105. Hydrocyanic
acid, 19. mercury, 172.
Metallic albummates, 172.
Methyl alcohol, 52, 259. Ni-
cotine, 91. Nitrobenzene,
42. Oxalic acid, 190. Pep-
per, 287. Phosphorus, 16.
Picrotoxin, 66. Potassium
chlorate, 194. Saponins,
221. Solanine, 226. Sul-
phurous acid, 188. Tin, 181.
Toxalbumins, 229. Ura-
nium, 1 80.
Tribromophenol, 28.
Trichlor-ethyl alcohol (see Alcohol,
trichlor-ethyl).
Trimethyl-amine, 211.
Trional, 203.
Triphenyl-methane, p-dihydroxy, 30.
Troches, Santonin, 291.
Tropidine, 106.
Tropine, 103, 106.
Turpentine, ozonized, Blood, 312.
Tyrosine, 17, 28.
U
Ulex europaeus, 205.
Ulexine (see Cytisine).
Uranium, 172, 180.
Urobilin, 17.
Urotheobromine, 300.
Vanadic-sulphuric acid test, Solanine,
226.
Van der Moer's test, Cytisine, 206.
Veratridine, 94.
Veratrine, 87, 93, 143. Extract, 87.
Veronal, 66, 79, 142.
Vitali's test, Alcohol, 51. Alkalies,
193. Atropine, 104. Blood,
313. Carbon disulphide, 49.
Veratrine, 96.
Vortmann's test, Hydrocyanic acid,
2.3-
W
Wangerin's test, Apomorphine, 128.
Narcotine, 115.
Warren's test, Papaverine, 217.
Weehuizen's test, Hydrocyanic acid,
24.
Weppen's test, Veratrine, 95.
White arsenic, 150.
Wine of opium, Morphine, 285.
Wine, Salicylic acid, 250. Saponins,
223.
Wormseed, Santonin, 289.
X
Xanthine bases, Urine, 84.
Xanthogenate test, Carbon disulphide,
47-
o-Xylene, 204.
Yellow phosphorus, 5.
Z
Zeisel's test, Colchicin, 69. Methoxyl,
101.
Zinc, 1 68, 1 80.
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